JP2014040419A - Pd-1, receptor of b7-4 and use of the same - Google Patents

Pd-1, receptor of b7-4 and use of the same Download PDF

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JP2014040419A
JP2014040419A JP2013172201A JP2013172201A JP2014040419A JP 2014040419 A JP2014040419 A JP 2014040419A JP 2013172201 A JP2013172201 A JP 2013172201A JP 2013172201 A JP2013172201 A JP 2013172201A JP 2014040419 A JP2014040419 A JP 2014040419A
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Clive Wood
クライブ・ウッド
Gordon J Freemann
ゴードン・ジェイ・フリーマン
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Dana Farber Cancer Institute Inc
デイナ ファーバー キャンサー インスティチュート,インコーポレイテッド
Genetics Inst Llc
ジェネティクス インスティテュート,エルエルシー
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Abstract

PROBLEM TO BE SOLVED: To provide a method to modulate immune response.SOLUTION: PD-1 polypeptide is identified as a receptor to B7-4. B7-4 can inhibit the activation of an immunocyte when it is bound to an inhibitory receptor on the immunocyte. Therefore, modulation of immune response is induced by modulating costimulatory or inhibitory signal in the immunocyte by providing an agent which modulates PD-1, B7-4 and interaction between B7-4 and PD-1.

Description

Government funding The study described herein was supported by AI39671, AI44690, CA84500, and AI41584 awarded by the National Institute of Health. Therefore, the US government may have certain rights in the invention.

Related Applications This application is a U.S. application filed on August 23, 1999. S. S. N. It claims priority to 60 / 150,390. This application is a U.S. application filed on Nov. 10, 1999. S. S. N. It also claims priority to 60/1664897. Both of these applications are hereby incorporated by reference.

BACKGROUND OF THE INVENTION In order for T cells to respond to foreign proteins, two signals must be provided to resting T lymphocytes by antigen presenting cells (APC) (Jenkins, M. and Schwartz, R (1987) J. Exp. Med. 165, 302-319; Mueller, DL, et al. (1990) J. Immunol. 144, 3701-3709). The first signal imparts specificity to the immune response and is transmitted through the T cell receptor (TCR) after recognition of the foreign antigenic peptide presented by the major histocompatibility complex (MHC). The second signal is called costimulation and induces T cells to proliferate and become functional (Lenschow et al. 1996. Annu. Rev. Immunol. 14: 233). Costimulation is not antigen specific, is not restricted by MHC, and is thought to be provided by one or more distinct cell surface molecules expressed by APC (Jenkins, MK, et al. 1988 J. Immunol. 140 , 3324-3330; Linsley, PS, et al. 1991 J. Exp. Med. 173 , 721-730; Gimmi, CD, et al., 1991 Proc. Natl. Acad. Sci. USA. 88 , 6575-6579; Young, JW, et al. 1992 J. Clin. Invest. 90 , 229-237; Koulova, L., et al. 1991 J. Exp. Med. 173 , 759-762; Reiser, H ., et al. 1992 Proc. Natl. Acad. Sci. USA. 89 , 271-275; van-Seventer, GA, et al. (1990) J. Immunol. 144 , 4579-4586; LaSalle, JM, et al , 1991 J. Immunol. 147 , 774-80; Dustin, MI, et al., 1989 J. Exp. Med. 169 , 503; Armitage, RJ, et al. 1992 Nature 357 , 80-82; Liu, Y , et al. 1992 J. Exp. Med. 175 , 437-445).
CD80 (B7-1) and CD86 (B7-2) proteins expressed on APCs are important costimulatory molecules (Freeman et al. 1991. J. Exp. Med. 174: 625; Freeman et al. 1989 J. Immunol. 143: 2714; Azuma et al. 1993 Nature 366: 76; Freeman et al. 1993. Science 262: 909). B7-2 appears to play an important role during the primary immune response, and B7-1 is upregulated late in the immune response to prolong the primary T cell response or to co-stimulate the secondary T cell response (Bluestone. 1995. Immunology. 2: 555).
CD28, one ligand to which B7-1 and B7-2 bind, is constitutively expressed on resting T cells, with increased expression after activation. Following signaling through the T cell receptor, CD28 and costimulatory signal transduction cooperate to induce T cell proliferation and IL-2 secretion (Linsley, PS, et al. 1991 J. Exp. Med. 173 , 721 -730; Gimmi, CD, et al. 1991 Proc. Natl. Acad. Sci. USA. 88 , 6575-6579; June, CH, et al. 1990 Immunol. Today. 11 , 211-6; Harding, FA, et al. 1992 Nature. 356 , 607-609). A second ligand called CTLA4 (CD152) is homologous to CD28 but is not expressed on resting T cells and appears after T cell activation (Brunet, JF, et al., 1987 Nature 328). , 267-270). In contrast to CD28, CTLA4 appears to be important in negative regulation of T cell responses (Waterhouse et al. 1995. Science 270: 985). While blocking of CTLA4 has been found to eliminate the inhibitory signal, CTLA4 aggregation has been found to provide an inhibitory signal that down-regulates T cell responses (Allison and Krummel. 1995. Science 270: 932). The B7 molecule has a higher affinity for CTLA4 than for CD28 (Linsley, PS et al. (1991) J. Exp. Med. 174: 561-569), B7-1 and B7-2 are CTLA4 molecules. It has been found to bind to different regions and have different binding kinetics for CTLA4 (Linsley et al. (1994) Immunity 1: 793). ICOS, a new molecule related to CD28 and CTLA4, has been identified and has a ligand that is a new member of the B7 family (Aicher A. et al. (2000) J. Immunol. 164: 4689-96; Mages HW et al. (2000) Eur. J. Immunol. 30: 1040-7; Brodie D. et al. (2000) Curr. Biol. 10: 333-6; Ling V. et al. (2000) J. Immunol 164: 1653-7; Yoshinaga SK et al. (1999) Nature 402: 827-32), which appears to be important in IL-10 production (Hutloff et al. (1999) Nature 397: 263; WO98 / 38216). ). If T cells are stimulated only through the T cell receptor without receiving additional costimulatory signals, they become non-responsive, anergic, or die, resulting in down-modulation of the immune response.
The importance of the B7: CD28 / CTLA4 costimulatory pathway has been demonstrated in vitro and also in an in vivo model system. Blocking this costimulatory pathway causes the development of antigen-specific resistance in murine and human systems (Harding, FA et al. (1992) Nature 356: 607-609; Lenschow, DJ et al. (1992) Science 257: 789-792; Turka, LA et al. (1992) Proc. Natl. Acad. Sci. USA 89: 11102-11105; Gimmi, CD et al. (1993) Proc. Natl. Acad. Sci. USA 90 : 6586-6590; Boussiotis, V. et al. (1993) J. Exp. Med. 178: 1753-1763). Conversely, B7 expression by B7-negative murine tumor cells induces specific immunity with tumor rejection mediated by T cells as well as protection against long-lasting tumor challenge (Chen, L. et al. (1992) Cell 71: 1093-1102; Townsend, SE and Allison, JP (1993) Science 259: 368-370; Baskar, S. et al. (1993) Proc. Natl. Acad. Sci. 90: 5687-5690 ). Therefore, manipulating the costimulatory pathway provides great potential for stimulating or suppressing immune responses in humans.

SUMMARY OF THE INVENTION The present invention is based, at least in part, on the finding that PD-1 is a receptor for the B7-4 molecule expressed on antigen presenting cells. PD-1, like CTLA4, transmits negative signals to immune cells. B7-4 molecules are expressed on the surface of antigen-presenting cells and can provide costimulatory signals to immune cells and transmit down-regulation signals to immune cells depending on the molecules that bind. Thus, modulating the interaction between PD-1, B7-4, and / or B7-4 and PD-1 results in modulation of the immune response.

Accordingly, in one aspect, the invention provides a method of modulating an immune response, comprising modulating an immune response by contacting an immune cell with an agent that modulates signaling through PD-1.
In one embodiment, the immune response is downregulated.
In another embodiment, it comprises an activating antibody that recognizes PD-1, a form of B7-4 that binds to an inhibitory receptor, and a small molecule that binds to PD-1. Signaling through PD-1 is stimulated with an agent selected from the group.
In one embodiment, the immune cells are selected from the group consisting of T cells, B cells and bone marrow cells.
In one embodiment, anergy is induced in immune cells.
In one embodiment, there is provided a method further characterized by contacting the immune cell with an additional agent that down regulates the immune response.
In one embodiment, the immune response is upregulated.
In one embodiment, an agent selected from the group consisting of a blocking antibody that recognizes PD-1, an inactivated form of B7-4, an antibody that recognizes B7-4, and a soluble form of PD-1 is used. Thus, signaling through PD-1 is inhibited.
In one embodiment, the contacting step is performed in vivo. In another embodiment, the contacting step is performed in vitro.

In another aspect, the present invention relates to a method of modulating the interaction of an inhibitory receptor on immune cells with B7-4, which comprises expressing an antigen presenting cell expressing B7-4 to B7-4. In contact with an agent selected from the group consisting of one form of PD-1, one form of PD-1, or an agent that modulates the interaction between B7-4 and PD-1, It is characterized by modulating its interaction with inhibitory receptors.
In one embodiment, the method is further characterized by contacting immune cells or antigen presenting cells with additional agents that modulate the immune response.
In one embodiment, the contacting step is performed in vivo. In another embodiment, the contacting step is performed in vitro.
In one embodiment, the immune cells are selected from the group consisting of T cells, B cells and bone marrow cells.

  In another aspect, the present invention relates to a method of inhibiting activity in immune cells via a non-apoptotic mechanism, the method comprising increasing PD-1 activity or expression in immune cells to activate immune cell activation. It is characterized by inhibiting.

In another aspect, the invention relates to a vaccine comprising an antigen and an agent that inhibits signaling through PD-1 in immune cells.
In another aspect, the invention relates to a composition comprising an antigen and an agent that promotes PD-1 mediated signaling in immune cells.

In another aspect, the present invention relates to a method of treating a subject having a condition that would benefit from up-regulation of the immune response, the method comprising immunizing the subject with an agent that inhibits signaling through PD-1. It is characterized in that it is administered to cells to treat conditions that would benefit from upregulation of the immune response.
In one embodiment, the agent comprises a soluble form of PD-1 or B7-4.
In one embodiment, the method is further characterized by administering to the subject a second agent that upregulates an immune response.
In one embodiment, the symptom is selected from the group consisting of a tumor, a neurological disease or an immunosuppressive disease.

In another aspect, the invention relates to a method of treating a subject having a condition that would benefit from down-regulation of the immune response, the method comprising immunizing the subject with an agent that stimulates signaling through PD-1. Administered to cells to treat conditions that would benefit from down-regulation of the immune response.
In one embodiment, the agent is selected from the group consisting of an antibody that stimulates signaling through PD-1, a bispecific antibody, and soluble B7-4.
In one embodiment, the method is further characterized by administering to the subject a second agent that down regulates the immune response.
In one embodiment, the symptom is selected from the group consisting of grafts, allergies, and autoimmune diseases.

  In another aspect, the invention relates to a cell-based assay for screening compounds that modulate the activity of B7-4 or PD-1, wherein the method comprises a B7-4 target molecule or a PD-1 target molecule Is characterized in that the cell expressing the is contacted with a test compound and then the ability of the test compound to modulate the activity of the B7-4 or PD-1 target molecule is characterized.

  In yet another aspect, the invention relates to a cell-free assay for screening compounds that modulate the binding of B7-4 or PD-1 to a target molecule, the method comprising a B7-4 or PD-1 protein. Or contacting the biologically active portion thereof with a test compound and then determining the ability of the test compound to bind to the B7-4 or PD-1 protein or biologically active portion thereof. And

FIG. 1 shows the nucleotide sequence encoding human secreted B7-4, B7-4S (SEQ ID NO: 1). FIG. 2 shows the nucleotide sequence encoding human secreted B7-4, B7-4M (SEQ ID NO: 3). FIG. 3 shows the amino acid sequence of human B7-4S (SEQ ID NO: 2), showing the signal, IgV, IgC, and hydrophilic tail domain. FIG. 4 shows the amino acid sequence of human B7-4M (SEQ ID NO: 4), showing the signal, IgV, IgC, and hydrophilic tail domain. FIG. 5 shows the nucleotide sequence of murine B7-4 (SEQ ID NO: 22). FIG. 5 shows the nucleotide sequence of murine B7-4 (SEQ ID NO: 22). FIG. 6 shows the amino acid sequence of murine B7-4 (SEQ ID NO: 23). FIG. 7 compares the human and murine B7-4 amino acid sequences. FIG. 8 shows the results of FACS analysis for binding of CD28Ig, CTLA4-Ig, and control Ig by COS cells transfected with B7-4M. FIG. 8 shows the results of FACS analysis for binding of IgG and murine ICOS-his fusion protein by COS cells transfected with B7-4M. FIG. 10 shows the results of FACS analysis for binding of IgM, BB1 and 133 antibodies to COS cells transfected with B7-4M. FIG. 11 shows that COS cells transfected with B7-4M (292) can costimulate T cell proliferation. FIG. 12 shows that COS cells transfected with B7-4M (292) can costimulate T cell proliferation. FIG. 13 shows the binding of PD-1 to COS cells transfected with B7-4. FIG. 5 shows the ability of added PD-1 and the inability of Flt4 to compete for PD-1 binding to COS cells transfected with B7-4M. FIG. 15 shows the ability of PD-1 to bind to CHO cells transfected with B7-4 as examined by flow cytometry. FIG. 16 shows the ability of PD-1 to bind to CHO cells transfected with B7-4 as determined by BIACORE analysis. FIG. 17 shows the ability of B7-4M to transmit negative signals to T cells. FIG. 18 shows inhibition of T cell proliferation and cytokine production in human T cells in the presence of B7-4. FIG. 19 shows that T cell receptor / B7-4 activation inhibits T cell proliferation in the presence of CD28 costimulation. FIG. 20 shows the binding of PD-1 to CHO cells expressing B7-4. The effect | action of B7-4 in CD28 signal inhibition is shown. FIG. 22 shows inhibition of cytokine production by the PD-1: B7-4 system as measured by cytokine ELISA. FIG. 5 shows inhibition of cytokine production by the PD-1: B7-4 pathway as measured by cytokine mRNA levels. FIG. 24 shows that the mechanism of action of the PD-1: B7-4 pathway is cell cycle arrest. FIG. 25 shows the ability of antibodies to B7-4 to inhibit the interaction between B7-4 and PD-1. FIG. 26 shows the ability of antibodies to PD-1 to inhibit the interaction between B7-4 and PD-1. FIG. 27 shows the ability of soluble B7-4Fc to exacerbate disease in an experimental murine autoimmune encephalomyelitis model.

Detailed Description of the Invention In addition to the previously characterized B lymphocyte activating antigens, such as B7-1 and B7-2, other antigens that modulate costimulation of immune cells are present on the surface of antigen presenting cells To do. For example, B7-4 polypeptides have been isolated from keratinocytes and placental cDNA libraries. B7-4 has also been found to costimulate or inhibit T cells.

  Immune cells have receptors that transmit activation signals. For example, T cells have a T cell receptor and a CD3 complex, B cells have a B cell receptor, and bone marrow cells have an Fc receptor. Furthermore, immune cells have receptors that transduce signals that provide costimulatory signals or that transduce signals that are mediated by receptors. For example, CD28 transmits a costimulatory signal to T cells. Following ligation of the T cell receptor, ligation of CD28 is e.g. upregulation of IL-2rα, IL-2rβ and IL-2rγ receptors, increased transcription of IL-2 messenger RNA, cytokines (IL-2, IFN) (Including γ, GM-CSF, and TNF-a) give rise to costimulatory signals characterized by increased gene expression. By transmitting a costimulatory signal, the cell undergoes the cell cycle to promote T cell proliferation (Greenfield et al. (1998) Crit. Rev. Immunol. 18: 389). Binding to receptors on T cells that transmit costimulatory signals to cells by B7 family molecules such as B7-4 (eg, ligation of costimulatory receptors to induce T cell cytokine secretion and / or proliferation). Causes costimulation. Thus, inhibiting the interaction between a B7 family molecule such as B7-4 and a receptor that transmits a costimulatory signal to immune cells causes a downregulation of the immune response and / or a special no response called anergy. . Inhibiting this interaction can be achieved, for example, by using antibodies to anti-CD28 Fab fragments, B-1, B7-2 and b / or B7-4, or B7 family member molecules as competitive inhibitors. This can be done by using a soluble form of the receptor that can bind (eg CTLA4Ig).

  Inhibitory receptors that bind to costimulatory molecules have also been identified on immune cells. Activation of CTLA4, for example, transmits a negative signal to T cells. Use of CTLA4 can inhibit IL-2 production and induce cell cycle arrest (Krummel and Allison (1996) J. Exp. Med. 183: 2553). Furthermore, CTLA4-deficient mice develop lymphoproliferative diseases (Tivol et al. (1995) Immunity 3: 541; Waterhouse et al. (1995) Science 270: 985). Blocking CTLA4 with an antibody can remove the inhibitory signal, but aggregation of CTLA4 with the antibody transmits the inhibitory signal. Thus, depending on the receptor to which the costimulatory molecule binds (eg, a costimulatory receptor such as CD28 or an inhibitory receptor such as CTLA4), certain B7 molecules may promote T cell costimulation or inhibition. .

  PD-1 is a molecular immunoglobulin member (Ishida et al. (1992) EMBO J. 11: 3887; Shinohara et al. (1994) Genomics 23: 704). PD-1 has already been identified by a subtractive cloning-based approach designed to identify molecules for programmed cell death (Ishida et al. (1992) EMBO J. 11: 3887-95; Woronicz et al (1995) Curr. Top. Microbiol. Immunol. 200: 137). PD-1 is thought to play a role in, for example, lymphocyte survival during clonal selection (Honjo (1992) Science 258: 591; Agatab et al. (1996) Int. Immunology. 8: 765; Nishimura et al. (1996) Int. Immunology 8: 773). PD-1 is also involved as a regulator of B cell responses (Nishimura (1998) Int. Immunology 10: 1563). Unlike CTLA4, which is found only on T cells, PD-1 is also found on B cells and bone marrow cells.

This finding that PD-1 binds to B7-4 positions PD-1 together with CTLA4 in the inhibitory receptor family. While the use of costimulatory receptors produces costimulatory signals in immune cells, the use of inhibitory receptors such as CTLA4 or PD-1 (eg, by cross-linking or aggregation) transmits inhibitory signals in immune cells. Cause down-regulation of immune cell responses and / or immune cell anergy. Inhibitory signal transduction causes down-modulation of the immune cell response (and causes down-modulation of the overall immune response), but prevention of inhibitory signals in immune cells (eg, non-activated antibodies against PD-1) Causes the up-modulation of the immune cell response (and causes up-modulation of the immune response).
The present invention relates to PD-1 activity and / or expression; agents useful to modulate the interaction of PD-1 with its native ligand (eg, B7-4); and PD-1 and its native Agents that modulate the immune response through modulation of their interactions with other ligands such as B7-4. Exemplary modulation agents used in these methods are further described below.

B7-4 and PD-1 nucleic acid and polypeptide molecules In one embodiment, modulation agents useful for modulating PD-1 activity and / or expression are B7-4 and / or PD-1 nucleic acid molecules, Preferably, it is a human B7-4 and / or PD-1 nucleic acid molecule.
In one embodiment, the isolated nucleic acid molecule of the invention encodes a eukaryotic B7-4 or PD-1 polypeptide. The B7-4 family of molecules shares many conserved regions including the signal domain, IgV domain and IgC domain. The IgV domain and IgCd main are recognized domain domains of the Ig superfamily members. These domains correspond to structural units with a unique folding pattern called Ig folds. The Ig fold is composed of two β sheets, each sheet consisting of an antiparallel β chain of 5-10 amino acids, a disulfide conserved between the two sheets in most if not all domains. There is a bond. The IgC domains of Ig, TCR and MHC molecules have the same type of sequence pattern and are referred to as the C1-set in the Ig superfamily. Other IgC domains are included in other sets. IgV domains also share sequence patterns and are called V set domains. The IgV domain is longer than the C-domain and forms an additional pair of β chains.
Two forms of human B7-4 molecule have been identified. One form is a naturally occurring B7-4 soluble polypeptide, ie, having a short hydrophilic domain and no transmembrane domain, referred to herein as B7-4S (shown in SEQ ID NO: 2). ). The second form is a cell binding polypeptide, ie, having a transmembrane domain and a cytoplasmic domain, referred to herein as B7-4M (shown in SEQ ID NO: 4).
The B7-4 protein contains a signal sequence, an IgV domain and an IgC domain. The signal sequence of SEQ ID NO: 2 is shown from approximately amino acid 1 to approximately amino acid 18. The signal sequence of SEQ ID NO: 4 is shown from about amino acid 1 to about amino acid 18. The IgV domain of SEQ ID NO: 2 is shown from about amino acid 19 to about amino acid 134, and the IgV domain of SEQ ID NO: 4 is shown from about amino acid 19 to about amino acid 134. The IgC domain of SEQ ID NO: 2 is shown from about amino acid 135 to about amino acid 227, and the IgC domain of SEQ ID NO: 4 is shown from about amino acid 135 to about amino acid 227. The hydrophilic tail of B7-4 shown in SEQ ID NO: 2 includes the hydrophilic tail shown from about amino acid 228 to about amino acid 245. The B7-4 polypeptide shown in SEQ ID NO: 4 has a transmembrane domain shown from about amino acid 239 to about amino acid 259 of SEQ ID NO: 4 and a cytoplasm shown from about amino acid 260 to about amino acid 290 of SEQ ID NO: 4. Includes domain.
A murine B7-4 molecule was also identified. The murine cDNA sequence is shown in FIG. 5, and the murine B7-4 amino acid sequence is shown in FIG. The invention also relates to these murine B7-4 sequences.

Herein, PD-1 has been identified as a receptor that binds to B7-4. The PD-1 molecule is a member of the immunoglobulin gene superfamily. PD-1 (Ishida et al. (1992) EMBO J. 11: 3887; Shinohara et al. (1994) Genomics 23: 704; US Pat. No. 5,698,520) is an extracellular region comprising an immunoglobulin superfamily domain, a membrane It has a transmembrane domain and an intracellular region (ITIM) that contains an immunoreceptor tyrosine-based inhibition motif. These characteristics also define a larger family of molecules called immunoinhibitory receptors, which include gp49B, PIR-B, and killer inhibitory receptors (KIRs) (Vivier and Daeron (1997). ) Immunol. Today 18: 286). The tyrosyl phosphorylated ITIM motif of these receptors is thought to interact with the SH2-domain, including phosphatases, to generate inhibitory signals. A subset of these immunoinhibitory receptors bind to MHC molecules, such as KIRs, and CTLA4 binds to B7-1 and B7-2. It has been proposed that there is a phylogenetic relationship between the MHC gene and the B7 gene (Henry et al. (1999) Immunol. Today 20 (6): 285-8).
The nucleotide sequence of PD-1 is shown in SEQ ID NOs: 10 and 11, and the amino acid sequence of PD-1 is shown in SEQ ID NO: 12 (Ishida et al. (1992) EMBO J. 11: 3887; Shinohara et al. (1994 Genoimics 23: 704; see US Pat. No. 5,698,520). PD-1 has already been identified using a subtractive cloning-based approach to select proteins involved in apoptotic cell death. PD-1 has been identified herein as a member of the CD28 / CTLA4 family of molecules based on its ability to bind B7-4. Similar to CTLA4, PD-1 is rapidly induced on the T cell surface in response to anti-CD3 (Agata et al. (1996) Int. Immunol. 8: 765). However, in contrast to CTLA4, PD-1 is also induced on the B-cell surface (in response to anti-IgM). PD-1 is also expressed on a subset of thymocytes and bone marrow cells (Agata et al., Supra; Nishimura et al. (1996) Int. Immunol. 8: 773). According to the present invention, B7-4 is identified as a ligand for PD-1.
Various aspects of the invention are described in further detail in the following subsections.

I. Definitions The term “immune cell” as used herein encompasses cells of hematopoietic origin that play a role in the immune response. Immune cells include lymphocytes such as B cells and T cells; natural killer cells; bone marrow cells such as monocytes, macrophages, eosinophils, mast cells, basophils and granulocytes.
As used herein, the term “T cell” includes CD4 + cells and CD8 + cells. The term “T cell” also includes T helper type 1 T cells and T helper type 2 T cells. The term “antigen presenting cell” refers to specialized antigen presenting cells (eg, B lymphocytes, monocytes, dendritic cells, Langerhans cells) as well as other antigen presenting cells (eg, keratinocytes, endothelial cells, astrocytes, fibers). Blast cells, oligodendrocytes).
The term “immune response” as used herein encompasses T cell mediated and / or B cell mediated immune responses that are affected by modulation of T cell costimulation. Typical immune responses include T cell responses, such as cytokine production, and cellular cytotoxicity. Furthermore, the term “immune response” encompasses immune responses indirectly triggered by T cell activation, such as antibody production (humoral response) and activation of cytokine responsive cells such as macrophages.
As used herein, the term “costimulatory receptor” encompasses receptors that transmit a costimulatory signal to an immune cell, eg, CD28. As used herein, the term “inhibitory receptor” includes receptors that transmit negative signals to immune cells (eg, CTLA4 or PD-1). An inhibitory signal may be generated that is converted by an inhibitory receptor even if the costimulatory receptor (such as CD28) is not present on immune cells, and thus it is an inhibitory receptor for costimulatory molecules. It is not just a function of competition between the body and costimulatory receptors (Fallarino et al. (1998) J. Exp. Med. 188: 205). Inhibitory signal transduction to immune cells can cause no response or anergy or programmed cell death in immune cells. Preferably, the transmission of inhibitory signals is one that operates via a mechanism that does not involve apoptosis. The term “apoptosis” herein includes programmed cell death that can be characterized using methods known in the art. Apoptotic cell death can be characterized, for example, by chromatin enrichment that maximizes cell contraction, membrane vacuolation and cell fragmentation. Cells undergoing apoptosis also exhibit a characteristic pattern of internucleosomal DNA cleavage.
Depending on the form of the B7-4 molecule that binds to the receptor, a signal can be transmitted (eg, by a multivalent form of the B7-4 molecule that causes cross-linking of the receptor) or, for example, to the receptor Either the signal can be inhibited by competing with the activated form of the B7-4 molecule for binding (eg, by the soluble monovalent form of the B7-4 molecule). However, there are examples where soluble molecules can be inhibitory. The effects of various modulation agents can be readily demonstrated by routine screening assays as described herein.

The term “costimulatory” herein for activated immune cells refers to a second non-activating signal (“costimulatory signal”) that is mediated by the receptor and induces proliferation or effector function. Including the ability of costimulatory molecules to provide For example, a costimulatory signal can cause cytokine secretion in a T cell that has received a signal mediated, for example, by a T cell receptor. For example, an immune cell that has received a signal mediated by a cellular receptor via an activating receptor is referred to herein as an “activated immune cell”.
As used herein, the term “activated receptor” encompasses an immune cell receptor that binds to an antigen, complexed antigen (eg, in the context of an MHC molecule), or binds to an antibody. Such activated receptors include T cell receptor (TCR), B cell receptor (BCR), cytokine receptor, LPS receptor, complement receptor, and Fc receptor.

For example, T cell receptors are present on T cells and bind to CD3 molecules. In the context of MHC molecules, T cell receptors are stimulated by antigens (as well as by polyclonal T cell activation reagents). T cell activation via TCR causes many changes, such as protein phosphorylation, changes in membrane lipids, changes in ion flux, cyclic nucleotides, changes in RNA transcription, changes in protein synthesis, and changes in cell volume.
B cell receptors are present on B cells. The B cell antigen receptor is a complex between membrane Ig (mIg) and other transmembrane polypeptides (eg, Igα and Igβ). The signal transduction function of mIg is triggered by cross-linking of the receptor molecule with oligomeric or multimeric antigens. B cells can also be activated by anti-immunoglobulin antibodies. With BCR activation, many changes occur in B cells, including tyrosine phosphorylation.
Fc receptors are found on many cells involved in the immune response. Fc receptors (FcRs) are cell surface receptors for the Fc portion of immunoglobulin molecules (Igs). Among the human FcRs identified so far, those that recognize IgG (named FcγR), those that recognize IgE (FcεRI), those that recognize IgA (Fcα), and those that recognize polymerized IgM / A (FcμαR). FcRs are found in the cell types described: FcεRI (mast cells), FcεRII (many leukocytes), FcαR (neutrophils), and FcμαR (granular epithelium, hepatocytes) (Hogg, N. (1988) Immunol. Today 9: 185-86). FcγRs that have been extensively studied are key to cellular immune defense and are factors that stimulate the release of hydrolytic enzymes involved in the development of inflammatory mediators and autoimmune diseases (Unkeless, JC et al. (1988). Annu. Rev. UImmunol. 6: 251-81). FcγRs provide an important link between effector cells and Ig-secreting lymphocytes. This is because macrophages / monocytes, polymorphonuclear leukocytes, and natural killer (NK) cell FcγRs confer elements of specific recognition mediated by IgG. Human leukocytes have at least three different receptors for IgG. They are hFcγRI (found on monocytes / macrophages), hFcγII (found on monocytes, neutrophils, eosinophils, platelets, possibly B cells, and K562 cell lines), and FcγIII (NK cells, Found on neutrophils, eosinophils, and macrophages).
For T cells, transmission of costimulatory signals to T cells uses a signaling pathway that is not inhibited by cyclosporin A. In addition, costimulatory signals can induce cytokine (eg, IL-2 and / or IL-10) secretion in T cells and / or induce no response to antigen, induce anergy, or in T cells. May interfere with induction of cell death.

As used herein, the term “inhibitory signal” refers to a signal transmitted through an inhibitory receptor (eg, CTLA4 or PD-1) for a molecule on an immune cell. Such signals antagonize signals through activating receptors (via TCR, CD3, BCR, or Fc molecules), eg, second messenger production inhibition; growth inhibition; effector function inhibition in immune cells, eg phagocytosis Reduced, decreased antibody production, reduced cellular cytotoxicity, inability to produce immune cell mediators (such as cytokines (eg, IL-2) and / or mediators of allergic responses); or may cause anergy.
As used herein, the term “no response” includes immune cell refractivity to stimuli, eg, stimuli mediated by activated receptors or cytokines. No response can occur, for example, by exposure to immunosuppressive agents or exposure to high doses of antigen. As used herein, the term “anergy” or “tolerance” includes bending to stimuli mediated by activated receptors. In general, such bending is antigen-specific and persists after cessation of tolerizing agent administration. For example, anergy in T cells (as opposed to unresponsiveness) is characterized by a lack of cytokine production, such as IL-2. T cell anergy occurs when a T cell is exposed to an antigen and receives an initial signal (signal mediated by a T cell receptor or CD-3) in the absence of a second signal (costimulatory signal) . Under these conditions, cell re-exposure to the same antigen (even if re-exposure occurs in the presence of a costimulatory molecule) does not result in cytokine production and thus does not proliferate. However, antigenic T cells have a response to irrelevant antigens and can proliferate when cultured with cytokines (eg, IL-2). For example, T cell anergy due to IL-2 production deficiency by T lymphocytes can be observed as measured by proliferation assays using ELISA or indicator cell lines. Alternatively, a reporter gene can be constructed. For example, anergic T cells are unable to initiate IL-2 gene transcription induced by heterologous promoters under the control of a 5 ′ IL-2 gene enhancer or by other-mer AP1 sequences found in the enhancer ( Kang et al. (1992) Science 257: 1134).

B7-4 proteins and nucleic acid molecules constitute a family of molecules with specific conserved structural and functional characteristics. Similarly, PD-1 proteins and nucleic acids are members of a family of molecules with conserved structural and functional characteristics. The term “family” when referring to proteins and nucleic acids refers to two or more proteins having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Or means a nucleic acid molecule. Such family members may be naturally occurring or non-naturally occurring, may be derived from the same species, or may be derived from different species. . For example, the family may contain a first protein of human origin as well as other different proteins of human origin, or may contain homologues of non-human origin. Family members may also have common functional characteristics. The B7-4 molecules described herein are members of the B7 family of molecules. As used herein, the term “B7 family” or “B7 molecule” refers to B7 polypeptides such as B7-1, B7-2, B7-3 (recognized by antibody BB-1), B7h (Swallow et al. ( 1999) Immunity 11: 423), and / or costimulatory molecules having sequence homology to B7-4. For example, when compared using NCBI's BLAST program using default parameters (Blosum62 matrix with gap penalities set at existence 11 and extension 1 (see http://www.ncbi.nlm.nih.gov)), human B7− 1 and B7-2 have about 26% amino acid identity.
Preferred B7 polypeptides are those that can provide costimulatory or inhibitory signals to immune cells, thereby promoting immune cell responses. For example, in binding to costimulatory receptors, for example when present in a soluble form, B7-4 can induce costimulation of immune cells or inhibit costimulation of immune cells. it can. When bound to an inhibitory receptor, the B7-4 molecule can transmit an inhibitory signal to immune cells. Preferred B7 family members include B7-1, B7-2, B7-3 (recognized by antibody BB-1), B7h and B7-4 and soluble fragments or derivatives thereof. In one embodiment, a member of the B7 family binds to one or more receptors on immune cells, such as CTLA4, CD28, ICOS, PD-1 and / or other receptors, depending on the receptor Have the ability to transmit inhibitory or costimulatory signals to immune cells, preferably T cells.
Preferred PD-1 molecules are those that, for example when present in soluble monomeric form, can transmit an inhibitory signal to immune cells, thereby inhibiting immune cell effector function, or immune cells Can be promoted (for example, by competitive inhibition). Preferred PD-1 family members are those that bind to one or more receptors on antigen presenting cells, such as B7-1, B7-2, B7-4, and / or other molecules.
Further, in one embodiment, a protein that is a member of a protein family is bound by an antibody raised against the protein of one or more other family members. For example, anti-BB-1 antibody recognizes the B7-4 molecule.

The term “activity” herein for a B7-4 or PD-1 polypeptide encompasses activities inherent in the structure of a B7-4 or PD-1 protein. The term “activity” with respect to B7-4 refers, for example, to the ability to modulate costimulatory stimulation of immune cells by modulating costimulatory signals in activated immune cells, or by using natural receptors on immune cells, for example. Includes the ability to modulate stimulation by modulating inhibitory signals in immune cells. When the activated form of the B7-4 molecule binds to a costimulatory receptor, a costimulatory signal is generated in the immune cell. When the activated form of B7-4 molecule binds to an inhibitory receptor, an inhibitory signal is generated in immune cells.
Modulation of costimulatory signals causes modulation of immune cell effector functions. Thus, the term “B7-4 activity” encompasses the ability of a B7-4 polypeptide to bind to its native receptor, the ability to modulate immune cell costimulatory or inhibitory signals, and the ability to modulate an immune response. To do.
With respect to PD-1, the term “activity” encompasses the ability of a PD-1 polypeptide to modulate an inhibitory signal in activated immune cells, for example by using native ligands on antigen presenting cells. PD-1 transmits an inhibitory signal to immune cells in a manner similar to CTLA4. Modulation of inhibitory signals in immune cells causes modulation of immune cell proliferation and / or cytokine secretion of immune cells. PD-1 can also modulate costimulatory signals by competing with costimulatory receptors for binding of B7 molecules. Thus, the term “PD-1 activity” encompasses the ability of a PD-1 polypeptide to bind to its native ligand, the ability to modulate immune cell costimulatory or inhibitory signals, and the ability to modulate an immune response. .

As used herein, the term “naturally occurring” nucleic acid molecule refers to an RNA or DNA molecule having a naturally occurring nucleotide sequence (eg, encoding a natural protein).
As used herein, the term “antisense” nucleic acid molecule is a nucleotide sequence complementary to a “sense” nucleic acid encoding a protein, eg, a nucleotide sequence complementary to the coding strand of a double-stranded cDNA molecule, complementary to an mRNA sequence. Or a nucleotide sequence complementary to the coding strand of a gene. Thus, an antisense nucleic acid molecule can hydrogen bond to a sense nucleic acid molecule.
As used herein, the term “coding region” refers to a region of a nucleotide sequence that includes codons translated into amino acid residues, and the term “non-coding sequence” refers to a region of a nucleotide sequence that is not translated into amino acids (eg, 5 ′ and 3 'Untranslated region'.
As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid molecule linked thereto. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, and additional DNA segments can be ligated into the viral genome in the vector. Certain vectors are capable of autonomous replication in a host cell in which they are integrated. Some are incorporated into the genome of a host cell after introduction into the host cell, thereby replicating with the host genome (eg, non-episomal mammalian vectors). Moreover, certain vectors may direct the expression of genes that are operably linked. Such vectors are referred to as “recombinant expression vectors” or simply “expression vectors” in the present invention. In general, expression vectors useful in recombinant DNA methods are often in the form of plasmids. In the present specification, “plasmid” and “vector” are mixed. This is because plasmids are the most commonly used form of vector. However, the present invention is intended to encompass other forms of such vectors that perform equivalent functions, such as viral vectors (eg, replication deficient retoviruses, adenoviruses and adeno-associated viruses).
As used herein, the term “host cell” refers to a cell into which a nucleic acid molecule of the invention has been introduced, such as a recombinant expression vector of the invention. The terms “host cell” and “recombinant host cell” are mixed. It should be understood that such terms refer to the progeny or potential progeny of such a cell as well as the particular subject cell. Certain modifications can result in successive generations, either due to mutations or environmental effects, and in fact such progeny are not identical to the parent cell, but are encompassed within the terminology herein. Is done.

As used herein, the term “transgenic” animal refers to a non-human animal, preferably a mammal, more preferably a mouse, in which one or more cells of the animal contain a “transgene”. The term “transgene” refers to DNA that is integrated into the genome of a cell from which the transgenic animal develops and remains in the genome of the mature animal, eg, one or more cell types of the transgenic animal. Or it directs the expression of the gene product encoded in the tissue.
As used herein, the term “homologous recombinant animal” refers to one type of transgenic non-human animal, preferably a mammal, more preferably a mouse, in which the endogenous gene and the animal cell prior to animal development. The endogenous gene is changed by homologous recombination with an exogenous DNA molecule introduced into (eg, an animal embryonic cell).
As used herein, the term “isolated protein” is substantially free of other proteins, cellular materials and media when isolated from cells or produced by recombinant DNA methods, or chemically When synthesized, it refers to a protein that does not contain chemical precursors or other chemical reagents. An “isolated” or “purified” protein or biologically active protein thereof is substantially a cellular material or other contaminating protein derived from a cell or tissue source from which a B7-4 or PD-1 protein is derived. In the case of chemical synthesis, it does not contain chemical precursors or other chemical reagents. The term “substantially free of cellular material” refers to a B7-4 or PD-1 protein from which B7-4 or PD-1 has been isolated or separated from the cellular components of the cell produced by recombinant methods. Means a formulation of In one embodiment, the term “substantially free of cellular material” refers to less than about 30% (by dry weight) of non-B7-4 or PD-1 protein (also referred to herein as “contaminating protein”). ), More preferably about 20% non-B7-4 or PD-1 protein, more preferably less than about 10% non-B7-4 or PD-1 protein, most preferably less than about 5% non-B7-4 or Includes B7-4 or PD-1 protein preparations with PD-1 protein. Also, if the B7-4 or PD-1 protein or biologically active portion thereof is produced by recombinant methods, preferably it is free of media, ie the media is the volume of the protein formulation. Less than about 20%, more preferably less than about 10%, and most preferably less than about 5%.
The term “substantially free of chemical precursors or other chemical reagents” refers to a preparation of B7-4 or PD-1 protein in which the protein is separated from the chemical precursors or chemical reagents used in protein synthesis. Include. In one embodiment, the term “substantially free of chemical precursors or other chemical reagents” includes less than about 30% (by dry weight) of chemical precursors or non-B7-4 or PD-1 chemical reagents. Preferably less than about 20% chemical precursor or non-B7-4 or PD-1 chemical reagent, more preferably less than about 10% chemical precursor or non-B7-4 or PD-1 chemical reagent, most preferably about 5 Include B7-4 or PD-1 protein formulations with less than 1% chemical precursors or non-B7-4 or PD-1 chemical reagents.

The term “antibody” herein also encompasses an “antigen-binding portion” of an antibody (or simply “a portion of an antibody”). As used herein, the term “antigen-binding portion” refers to a fragment of one or more antibodies that retain specific binding to an antigen (eg, B7-4). It has been shown that the antigen-binding function of an antibody can function with fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody are: (i) a unitary fragment consisting of a Fab fragment, VL, VH, CL and CH1 domains; (ii) linked by a disulfide bridge at the hinge region. F (ab ′) 2 fragment, which is a bivalent fragment consisting of two Fab fragments ;; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the single arm VL and VH domains of the antibody (V) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341 : 544-546); and (vi) an isolated complementarity determining region (CDR). In addition, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but these can be joined by a synthetic linker that can be formed as a single protein chain using recombinant methods, the VL and VH regions. Are paired and are known as unit cost molecules (single chain Fv (scFv): eg Bird et al. (1988) Science 242 : 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85 : 5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16: 778). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. In order to obtain expression vectors that fully encode IgG molecules or other isomers, any of the VH and VL sequences of a particular scFv can be ligated to human immunoglobulin constant region cDNA or genomic sequences. VH and Vl can also be used to purify Fab, Fv or other immunoglobulin fragments using either protein chemistry or recombinant DNA methods. Also included are other forms of single chain antibodies such as diabodies. Diabodies are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but they are so short that they pair between two domains on the same chain. Can be used to force the domain to pair with the complementary domain of the other chain, forming two antigen-binding sites (eg, Holliger P., et al. al. (1993) Proc. Natl. Acad. Sci. USA 90 : 6444-6448; Poljak, RJ, et al. (1994) Structure 2 : 1121-1123).

Furthermore, an antibody or antigen-binding portion thereof is part of a larger immunoadhesion molecule and can be formed by covalent or non-covalent binding of an antibody or portion of an antibody to one or more proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin core region to create a tetrameric scFv molecule (Kipriyanov, SM et al. (1995) Human Antibodies and Hybridomas 6: 93-101) and divalent and biotinylated scFv Includes the use of cysteine residues to make the molecule, a labeled peptide and a C-terminal polyhistidine tag (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31 : 1047-1058). Portions of antibodies, such as Fab and F (ab ′) 2 fragments, can be produced from intact antibodies using conventional techniques such as papain and pepsin digestion of the intact antibodies. In addition, antibodies, portions of antibodies, and immunoadhesion molecules can be obtained using standard recombinant DNA methods described herein.
An antibody may be polyclonal or monoclonal; heterologous, homologous or syngeneic; or a modified form thereof, eg, humanized, chimeric, etc. Preferably, the antibodies of the invention bind specifically or substantially specifically to the B7-4 molecule. As used herein, the terms “monoclonal antibody” and “monoclonal antibody composition” refer to a population of antibody molecules comprising only one of the antigen binding sites that may immunoreact with a particular epitope of an antigen. In contrast, the terms “polyclonal antibody” and “polyclonal antibody composition” refer to a population of antibody molecules comprising a variety of antigen binding sites that may interact with a particular antigen. A monoclonal antibody composition typically exhibits single binding to a particular antigen with which it immunoreacts.

The term “humanized antibody” herein includes antibodies made by non-human cells that have variable and constant regions that are altered into more elaborate pseudo-antibodies made by human cells. For example, by altering non-human antibody amino acid sequences to incorporate amino acids found in human germline immunoglobulin sequences. Humanized antibodies of the present invention can be produced by, for example, amino acid residues that are not encoded by human germline immunoglobulin sequences in the CDRs (eg, random or site-specific mutants in vitro or somatic mutants in vivo. Derived variants). The term “humanized antibody” as used herein also encompasses antibodies in which CDR sequences derived from other mammalian species, eg, mouse embryo cell lines, are grafted onto human framework sequences.
As used herein, the term “isolated antibody” refers to an antibody that is substantially free of other antibodies having different antigen specificities (eg, an isolated antibody that specifically binds to B7-4 is B7-4). Substantially free of antibodies that specifically bind to other antigens). Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

As defined by the genetic code (below), there is a known and clear match between the amino acid sequence of a particular protein and the nucleotide sequence that can encode the protein. Similarly, there is a known and clear match between the nucleotide sequence of a particular nucleic acid molecule and the amino acid sequence encoded by that nucleic acid molecule, as defined by the genetic code.
An important and well-known feature of the genetic code is its duplication, so one or more coding nucleotide triplets can be used for most of the amino acids used to make proteins (top). Thus, many of the different nucleotide sequences can encode a given amino acid sequence. As a result, all living organisms produce similar amino acid sequences (though some organisms can be translated into some sequences more effectively than others), such nucleotide sequences are functionally It is considered equivalent. In addition, sometimes purine or pyrimidine methylated variants can be found at specific nucleotide sequences. Such methylation does not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.
In view of the above, the nucleotide sequence (or part thereof) of the DNA or RNA molecule encoding B7-4 or PD-1 herein is the genetic code for the DNA or RNA molecule to the amino acid sequence. It can be used for translation to derive B7-4 or PD-1 molecular amino acid sequences. Similarly, for B7-4 or PD-1-amino acid sequences, the corresponding nucleotide sequence capable of encoding B7-4 or PD-1 protein can be deduced from the genetic code (due to its degeneracy, either Resulting in a plurality of nucleic acid sequences for a particular amino acid sequence). Accordingly, the description and / or disclosure of a B7-4 or PD-1 nucleotide sequence herein also includes the description and / or disclosure of an amino acid sequence encoded by the nucleotide sequence. Similarly, the description and / or disclosure of a B7-4 or PD-1 amino acid sequence herein should also be considered to include the description and / or disclosure of all possible nucleotide sequences that can encode the amino acid sequence. It is.

II. In one embodiment of the isolated nucleic acid molecule, the modulation agent used in the claimed method includes an isolated nucleic acid molecule encoding a B7-4 or PD-1 protein or biological portion thereof. Also, a nucleic acid fragment sufficient for use as a hybridization probe to identify a B7-4 or PD-1-encoding nucleic acid (eg, B7-4 or PD-1 mRNA), and a B7-4 or PD-1 nucleic acid Also provided are fragments for use as PCR primers for amplification or mutation of molecules. As used herein, the term “nucleic acid molecule” includes DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA), where the DNA or RNA analog is generated using nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.

An “isolated” nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. For example, with respect to genomic DNA, the term “isolated” encompasses nucleic acid molecules from which the genomic DNA has been separated from naturally associated chromosomes. Preferably, an “isolated” nucleic acid molecule comprises a sequence that is naturally adjacent to the nucleic acid molecule in the genomic DNA of the organism that delivers the nucleic acid molecule (ie, the sequences located at the 5 ′ and 3 ′ ends of the nucleic acid). Not included. For example, in various embodiments, an isolated B7-4 or PD-1 nucleic acid molecule is about 5 kb, 4 kb, 3 kb, 2 kb, inherently adjacent to the nucleic acid molecule in the genomic DNA of the cell delivering the nucleic acid. It can comprise a nucleotide sequence of less than 1 kb, 0.5 kb or 0.1 kb. In addition, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially separated from other cellular material or medium when produced by recombinant methods, or is chemically precursor or otherwise when chemically synthesized. The chemical substance may be substantially not included. However, an “isolated” B7-4 or PD-1 nucleic acid molecule normally binds to other nucleotide sequences that are not adjacent to B7-4 or PD-1 in genomic DNA (eg, B7-4 or PD- A single nucleotide sequence can be linked to a vector sequence). In certain preferred embodiments, also “isolated” nucleic acid molecules, such as cDNA molecules, may be free of other cellular material. However, it is not necessary for the B7-4 or PD-1 nucleic acid molecule to be free of other cellular material to be considered “isolated” (eg, isolated from other mammalian DNA, bacterial cells B7-4 or PD-1 DNA molecules inserted into are still considered “isolated”).
Nucleic acid molecules of the invention, eg, nucleic acid molecules having the nucleotide sequence of SEQ ID NO: 1, 3, 10, or 11, or portions thereof, use standard molecular biology techniques and the sequence information provided herein. Can be isolated. For example, using all or part of the nucleic acid sequence of SEQ ID NO: 1, 3, 10 or 11 as a hybridization probe, B7-4 or PD-1 nucleic acid using standard hybridization and cloning methods. Molecules can be separated (eg, as described in Sambrook, J. et al. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
In addition, nucleic acid molecules encompassing all or part of SEQ ID NO: 1, 3, 10 or 11 use synthetic oligonucleotide primers designed based on each of the sequences of SEQ ID NO: 1, 3, 10 or 11. Can be isolated by polymerase chain reaction (PCR).

The nucleic acids of the invention can be amplified by standard PCR amplification methods using cDNA, mRNA or the respective genomic DNA as a template and special oligonucleotide primers. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. In addition, oligonucleotides corresponding to B7-4 or PD-1 nucleotide sequences can be produced, for example, by standard synthetic methods using automated DNA synthesizers.
In a preferred embodiment, the isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 1, 3, 10, or 11.
In other preferred embodiments, the isolated nucleic acid molecules of the invention include nucleic acid molecules that are the complement of the nucleotide sequence set forth in SEQ ID NO: 1, 3, 10, or 11 or a portion of any nucleotide sequence. . The nucleic acid molecule complementary to the nucleotide sequence shown in SEQ ID NO: 1, 3, 10 or 11 is one that is sufficiently complementary to the nucleotide sequence shown in each of SEQ ID NO: 1, 3, 10 or 11 Thus, it can hybridize to the nucleotide sequence shown in each of SEQ ID NOs: 1, 3, 10 or 11 and thus form a stable duplex.
In yet another preferred embodiment, the isolated nucleic acid molecule of the invention has at least about 50% of the nucleotide sequence set forth in SEQ ID NO: 1, 3, 10, or 11 or a portion of any of these nucleotide sequences. , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more nucleotide sequences that match.

  Furthermore, the nucleic acid molecule of the present invention can comprise only a part of the nucleic acid sequence of SEQ ID NO: 1, 3, 10 or 11, for example a fragment or B7-4 or PD- that can be used as a probe or primer. A fragment encoding the biological portion of a protein can be included. Nucleotide sequences determined from cloning of the B7-4 or PD-1 gene include B7-4 or PD-1 family homologs obtained from other species, and identification of other B7-4 or PD-1 family members and Allowing the generation of probes or primers designed for use in cloning. The probe / primer typically comprises a substantially preferred oligonucleotide. The oligonucleotide is typically a sense sequence of SEQ ID NO: 1, 3, 10 or 11 or a naturally occurring allelic variant of SEQ ID NO: 1, 3, 10 or 11 under stringent conditions. Or nucleotides that hybridize with at least about 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65 or 75 consecutive nucleotides of the mutant. Contains the region of the sequence. In typical embodiments, the nucleic acid molecules of the invention are at least 350, 400, 450, 500, 550, 600, 650, 700, 750 or 800 nucleotides in length and under stringent hybridization conditions, It includes a nucleotide sequence that hybridizes to the nucleic acid molecule sequence of SEQ ID NO: 1, 3, 10, or 11.

In other embodiments, the second nucleic acid molecule comprises at least about 500, 600, 700, 800, 900, or 1000 contiguous nucleotides of SEQ ID NO: 1, 3, 10, or 11.
In one embodiment, for example, a nucleic acid molecule of the invention used as a probe is of SEQ ID NO: 1 obtained from about nucleotides 815 to about 850 of SEQ ID NO: 1 or nucleotides 320 to 856 of SEQ ID NO: 1. Does not include some. In other embodiments, the nucleic acid molecule of the invention is SEQ ID NO: 3 derived from nucleotides approximately 314-734 or nucleotides approximately 835 to approximately 860 or nucleotides approximately 1085 to approximately 1104 or nucleotides approximately 1286 to approximately 1536 of SEQ ID NO: 3. 3 is not included.
In one embodiment, the nucleic acid molecule of the invention comprises at least about 500 contiguous nucleotides of SEQ ID NO: 1 or SEQ ID NO: 3. In preferred embodiments, the nucleic acid molecule of the invention comprises at least about 600, at least about 700, at least about 800, at least about 900 or at least 950 contiguous nucleotides of SEQ ID NO: 1 or about 1000 contiguous nucleotides of SEQ ID NO: 3. including. In other embodiments, the nucleic acid molecules of the invention comprise at least about 1500 or 1550 nucleotides of SEQ ID NO: 3.

Preferably, an isolated nucleic acid molecule of the invention comprises at least a portion of the coding region of SEQ ID NO: 1 (shown at nucleotides 59-793) or SEQ ID NO: 3 (shown at nucleotides 53-922). In other embodiments, the B7-4 nucleic acid molecule comprises about nucleotide 1 to nucleotide 319 of SEQ ID NO: 1. In other embodiments, the B7-4 nucleic acid molecule comprises from about nucleotide 855 to about nucleotide 968 of SEQ ID NO: 1. In other embodiments, the B7-4 nucleic acid molecule comprises about nucleotide 1 to nucleotide 314 of SEQ ID NO: 3. In other embodiments, the B7-4 nucleic acid molecule comprises from about nucleotide 955 to about nucleotide 1285 of SEQ ID NO: 3. In other embodiments, the B7-4 nucleic acid molecule comprises approximately nucleotides 1535 to approximately 1552 of SEQ ID NO: 3.
In other embodiments, the nucleic acid molecules of the invention have at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000 of SEQ ID NO: 1 or SEQ ID NO: 3. It has at least 70% identity, more preferably 80% identity, and even more preferably 90% identity with a nucleic acid molecule comprising nucleotides.
Probes based on B7-4 or PD-1 nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises an attached label group, for example, the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as part of a diagnostic test kit for the identification of cells or tissues that misexpress B7-4 or PD-1 protein in B7-4 or PD-1 in a sample of cells obtained from a subject. It can be used by measuring the level of the coding nucleic acid, for example, detecting B7-4 or PD-1 mRNA levels, or determining whether the genomic B7-4 or PD-1 gene is altered or deleted.

The nucleic acid fragment encoding “the biologically active portion of B7-4 or PD-1 protein” is B7-4 or PD-1 biological activity (the biological activity of B7-4 or PD-1 protein is And express the encoded portion of the B7-4 or PD-1 protein (eg, by recombinant expression in vitro), and of the B7-4 or PD-1 protein It can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO: 1, 3, 10, or 11 that encodes a polypeptide that evaluates the encoded portion of activity.
B7-4 or PD-1 which differs from SEQ ID NO: 1, 3, 10 or 11 due to the degeneracy of the genetic code but which is identical to that encoded by SEQ ID NO: 1, 3, 10 or 11 Nucleic acid molecules encoding proteins are encompassed by the present invention. Thus, in other embodiments, an isolated nucleic acid molecule of the invention has a nucleotide sequence that encodes a protein having the amino acid sequence set forth in SEQ ID NO: 2, 4, or 12. In other embodiments, the isolated nucleic acid molecules of the invention have a nucleotide sequence that encodes a B7-4 or PD-1 protein.
In addition to the B7-4 or PD-1 nucleotide sequence shown in SEQ ID NO: 1, 3, 10 or 11, a DNA sequence polymorphism leading to a change in the amino acid sequence of the B7-4 or PD-1 protein is It will be understood by those skilled in the art that, for example, it can be present in a human population). Such genomic polymorphisms of the B7-4 or PD-1 gene can exist in individuals in the population due to natural allelic variation. The terms “gene” and “recombinant gene” herein include an open reading frame encoding a B7-4 or PD-1 protein, preferably a mammalian B7-4 or PD-1 protein. Furthermore, it refers to a nucleic acid molecule that can include non-coding regulatory sequences and introns. Such natural allelic variations include both functional and non-functional B7-4 or PD-1 proteins, typically one in the nucleotide sequence of the B7-4 or PD-1 gene. Can cause ˜5% mutation. Such nucleotide variations and resulting amino acid polymorphisms in the B7-4 or PD-1 gene obtained as a result of natural allelic variation and which do not alter the functional activity of the B7-4 or PD-1 protein are: It is included in the scope of the present invention.
In addition, nucleic acid molecules that encode other B7-4 or PD-1 family members but have a nucleotide sequence that differs from the B7-4 or PD-1 family sequence of SEQ ID NO: 1, 3, 10 or 11 Are included in the present invention. For example, other B7-4 or PD-1 cDNAs can be identified based on the nucleotide sequence of human B7-4 or PD-1. Further, a nucleic acid molecule encoding a B7-4 or PD-1 protein obtained from a different species, but having a nucleotide sequence different from the B7-4 or PD-1 sequence of SEQ ID NO: 1, 3, 10 or 11 Is included in the scope of the present invention. For example, mouse B7-4 or PD-1 cDNA can be identified based on the nucleotide sequence of the human B7-4 or PD-1 molecule.

  Nucleic acid molecules corresponding to natural allelic variants and homologues of the B7-4 or PD-1 cDNA of the present invention are based on homology to the B7-4 or PD-1 nucleic acids disclosed herein. The cDNA disclosed herein or a portion thereof can be isolated as a hybridization probe by a conventional hybridization method. For example, B7-4 or PD-1 DNA can be isolated from a human genomic DNA library using all or part of SEQ ID NO: 1, 3, 10 or 11 as a hybridization probe and standard hybridization methods. (Eg, as described in Sambrook, J., et al. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). In addition, nucleic acid molecules comprising all or part of the B7-4 or PD-1 gene can be polymerase chain reaction using oligonucleotide primers designed based on the sequence of SEQ ID NO: 1, 3, 10, or 11. Can be isolated. For example, mRNA can be isolated from cells (eg, according to The guanidinium-thiocyanate extraction procedure of Chirgwin et al. (1979) Biochemistry 18: 5294-5299), and cDNA can be prepared using reverse transcription (eg, Moloney). MLV reverse transcriptase, available from Gibco / BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, FL). Synthetic oligonucleotide primers for PCR amplification can be designed based on the nucleotide sequence shown in SEQ ID NO: 1, 3, 10 or 11. The nucleic acid molecules of the invention can be amplified by standard PCR amplification methods using cDNA as the template, or alternatively genomic DNA, and appropriate oligonucleotide primers. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to B7-4 or PD-1 nucleotide sequences can be prepared by standard synthetic methods, eg, using an automated DNA synthesizer.

  In other embodiments, an isolated nucleic acid molecule of the invention is at least 15, 20, 25, 30 or more nucleotides in length and, under stringent conditions, SEQ ID NO: 1, 3, 10, or Hybridizes with a nucleic acid molecule comprising 11 nucleotide sequences. In other embodiments, the nucleic acid molecule is at least 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550 or 600 nucleotides in length. The term “hybridization under stringent conditions” herein refers to hybridization as well as nucleotide sequences that are typically hybridized to each other with at least 30%, 40%, 50% or 60% homology to each other. The conditions for cleaning as it is will be described. Preferably, conditions such that the sequences typically remain hybridized to each other with at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% homology to each other It is. Such stringent end conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1.-6.3.6. A preferred non-limiting example of stringent hybridization conditions is hybridization in 6X sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by 0.2X SSC at 50-65 ° C, 0.1 One or more washes with% SDS. Preferably, an isolated nucleic acid molecule of the invention that hybridizes with the sequence of SEQ ID NO: 1, 3, 10, or 11 under stringent conditions corresponds to a naturally occurring nucleic acid molecule.

As used herein, the term “naturally occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (eg, encodes a natural protein). In addition to the B7-4 or PD-1 nucleotide sequences shown in SEQ ID NOs: 1, 3, 10 and 11, DNA sequence polymorphisms leading to minor changes in the nucleotide or amino acid sequence of B7-4 or PD-1 are: It will be appreciated by those skilled in the art that they can exist in a population. Such genomic polymorphisms in the B7-4 or PD-1 gene can exist in individuals in the population due to natural allelic variation. Such natural allelic variations can typically give 1-2% variation in the nucleotide sequence of the gene. Such nucleotide variants and the resulting amino acid polymorphisms in B7-4 or PD-1 obtained as a result of natural allelic variation and not altering the functional activity of the B7-4 or PD-1 polypeptide are It is included in the scope of the invention.
In addition to naturally occurring allelic variants of B7-4 or PD-1 that can be present in a population, those skilled in the art can also, for example, mutate to the nucleotide sequence of SEQ ID NO: 1, 3, 10 or 11 It will be appreciated that minor changes can be introduced, and thus changes in the amino acid sequence of the encoded protein can be induced without altering the functional activity of the B7-4 or PD-1 protein. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made with the sequence of SEQ ID NO: 1, 3, 10 or 11. A “non-essential” amino acid residue may be a wild type sequence of a B7-4 nucleic acid molecule (eg, SEQ ID NO: 1, 3, 10, or 11) without altering the functional activity of the B7-4 or PD-1 molecule. Residues that can be changed from Preferably, B7-4 is bound to a receptor or PD-1 is bound to a natural ligand (eg, identified using an alanine scanning mutagenesis screen assay or other recognized screen assay). Residues in the extracellular fluid domain of B7-4 or PD-1 that are considered necessary for this are not changed. With respect to the B7-4 molecule, representative residues that are non-essential and thus susceptible to substitution are subjected to a side-by-side comparison of the amino acid sequence of the B7 family member (or B7-4 family member) to determine those that are not conserved. Can be identified by those skilled in the art. Such residues are not conserved and tend to be more easily substituted.

Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding B7-4 or PD-1 proteins that contain changes in amino acid residues that are not essential for B7-4 or PD-1 activity. Such a B7-4 or PD-1 protein differs from the amino acid sequence obtained from SEQ ID NO: 2, 4 or 12, but still maintains the intrinsic B7-4 activity or in the case of PD-1. Maintains the ability to bind to B7-4. An isolated nucleic acid molecule encoding a non-natural variant of a B7-4 or PD-1 protein is SEQ ID NO: so that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. One or more nucleotide substitutions, additions or deletions may be introduced into the 1, 3, 10 or 11 nucleotide sequences; Mutations can be introduced into SEQ ID NOs: 1, 3, 10 or 11 by standard methods such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions occur at one or more non-essential amino acid residues. “Conservative amino acid substitution” is one method of replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art and include basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid), uncharged polar side Chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta branched side chains (eg, Threonine, valine, isoleucine), and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Thus, preferably, a nonessential amino acid residue in B7-4 or PD-1 is replaced with another amino acid residue in a similar side chain family.
Alternatively, in other embodiments, the mutant is randomly derived with all or part of the B7-4 or PD-1 coding sequence, eg, by saturation mutagenesis, and the resulting mutant is DNA The ability to bind to and / or activate transcription can be screened to identify mutants that retain functional activity. Following mutagenesis, the encoded B7-4 or PD-1 mutein can be expressed in host cells by recombinant methods, and the functional activity of the mutein assesses B7-4 or PD-1 activity. Can be determined using analysis available in the art.

Accordingly, another aspect of the invention relates to nucleic acid molecules encoding B7-4 or PD-1 proteins that contain changes in amino acid residues that are not essential for activation.
Yet another aspect of the invention relates to an isolated nucleic acid molecule encoding a B7-4 or PD-1 fusion protein. At least a first nucleotide encoding a B7-4 or PD-1 protein, polypeptide or peptide operably linked to a second nucleotide sequence encoding a B7-4 or PD-1 protein, polypeptide or peptide Such nucleic acid molecules containing sequences can be prepared by standard recombinant DNA methods.
In a preferred embodiment, the mutant B7-4 protein is: 1) costimulation of activated immune cell proliferation and / or effector function (or inhibition of costimulation, eg, in soluble form); 2) Binding to anti-B7 family- or anti-B7-4-antibody; and / or 3) B7-4 (eg PD-1) may be assayed for its ability to bind to a natural receptor.
In a preferred embodiment, the mutant PD-1 protein is: 1) inhibition of co-stimulation of activated immune cell proliferation and / or effector function (eg, in soluble form); 2) anti-PD-1 antibody And / or 3) can be assayed for the ability of PD-1 to bind to a natural ligand (eg, B7-4).

  In addition to the nucleic acid molecule encoding the B7-4 or PD-1 protein, an antisense isolated nucleic acid molecule can be used as a modulation agent. An “antisense” nucleic acid comprises a nucleotide sequence complementary to a “sense” nucleic acid encoding a protein, eg, a nucleotide sequence complementary to the coding strand of a double-stranded cDNA molecule, or a nucleotide sequence complementary to an mRNA sequence. . Thus, an antisense nucleic acid can hydrogen bond with a sense nucleic acid. An antisense nucleic acid can be complementary to the entire B7-4 or PD-1 coding strand, or only to its protein. In one embodiment, the antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence encoding B7-4 or PD-1. The term “coding region” refers to a region of a nucleotide sequence that includes codons translated into amino acid residues. In other embodiments, the antisense nucleic acid molecule is antisense to a “non-coding region” of the coding strand of a nucleotide sequence encoding B7-4 or PD-1. The term “uncoding region” refers to 5 ′ and 3 ′ sequences that flank the coding region that are not translated into amino acids (eg, also refers to 5 ′ and 3 ′ untranslated regions).

  Once the coding strand sequences encoding B7-4 or PD-1 disclosed herein are obtained, the antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the complete coding region of B7-4 or PD-1 mRNA, but more preferably only the protein in the coding or non-coding region of B7-4 or PD-1 mRNA. Oligonucleotide. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of B7-4 or PD-1 mRNA. Antisense oligonucleotides can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. The antisense nucleic acids of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using processes known in the art. For example, antisense nucleic acid molecules (eg, antisense nucleotides) increase the biological stability of naturally occurring nucleotides or molecules and the physical stability of the duplex formed between the antisense and sense nucleic acids. Can be chemically synthesized using a variety of modified nucleotides designed to increase, for example, phosphorothioate derivatives and acridine substituted nucleotides. Examples of modified nucleotides that can be used to generate antisense nucleic acids are 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) Uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosilk eosin, inosine, N6-isopentenyladecine, 1-methylguanine, 1-methyllinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2 − Ouracil, beta-D-mannosilk eosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wivetoxosin, pseudouracil, queosin, 2 -Thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil , 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine. Instead, the antisense nucleic acid subclons the nucleic acid into an antisense orientation (ie, RNA transcribed from the inserted nucleic acid is in the antisense orientation to the relevant target nucleic acid, which is further described in the subsection below). It can be produced biologically using expression vectors.

  Antisense nucleic acid molecules of the invention are typically administered to a subject or produced in situ, which hybridize or encode cellular mRNA and / or B7-4 or PD-1 protein. It binds to genomic DNA and thus inhibits protein expression, for example, by inhibiting transcription and / or translation. Hybridization can form stable duplexes by conserved nucleotide complementarity or, for example, in the case of antisense nucleic acid molecules that bind to DNA duplexes, by special interactions at the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, a receptor expressed on the selected cell surface, such as by binding an antisense nucleic acid molecule to a peptide or antibody that binds to a cell surface receptor or antigen. It can be specifically modified to bind to the antigen. Antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. In order to achieve sufficient intracellular concentrations of the antisense molecule, a vector structure in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter is preferred.

In a further embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. α-anomeric nucleic acid molecules form specific double-strand hybrids with complementary RNA, where the strands extend parallel to each other as opposed to normal β-units (Gaultier et al. (1987) Nucleic Acids. Res. 15: 6625-6641). Antisense nucleic acid molecules can also include 2'-o-methyl ribonucleotides (Inoue et al. (1987) Nucleic Acids Res. 15: 6431-6148) or chimeric RNA-DNA analogs. (Inoue et al. (1987) FEBS Lett. 215: 327-330).
In still other embodiments, the antisense nucleic acid molecule of the invention is a ribozyme. Ribozymes are catalytic RNA molecules having ribonuclease activity that can cleave single-stranded nucleic acid molecules such as mRNA having regions complementary thereto. Thus, ribozymes (eg, hammerhead ribozymes (described in Haseloff and Gerlach (1988) Nature 334: 585-591)) can catalytically cleave B7-4 or PD-1 mRNA transcripts, and thus Ribozymes having specificity for B7-4 or PD-1-encoding nucleic acids can be used to inhibit translation of B7-4 or PD-1 mRNA. 1 Designed based on the nucleotide sequence of cRNA (ie, SEQ ID NO: 1, 3, 10 or 11. For example, Tetrahymena L-19 IVS RNA derivatives have an active site nucleotide sequence of B7-4 or PD- 1-coding mRNA can be constructed to be complementary to a nucleotide sequence that is cleaved into, eg, Cech et al. And US Pat. No. 5,116,742 to Cech et al. Alternatively, B7-4 or PD-1 mRNA is a catalyst having specific ribonuclease activity from a pool of RNA molecules. Can be used to select RNA, see, for example, Bartel, D. and Szostak, JW (1993) Science 261: 1411-1418.

Instead, B7-4 or PD-1 gene expression is a nucleotide sequence complementary to the regulatory region of B7-4 or PD-1 (eg, B7-4 or PD-1 promoter and / or enhancer). To form a triple helix structure that prevents transcription of the B7-4 or PD-1 gene in the target cell. In general, Helene, C. (1991) Anticancer Drug Des. 6 (6): 569-84; Helene, C. et al (1992) Ann. NY Acad. Sci. 660: 27-36; and Maher, LJ (1992) Bioessays 14 (12): 807-15.
In still other embodiments, the B7-4 or PD-1 nucleic acid molecules of the invention can be modified at the base, sugar, or phosphate backbone, eg, improving stability, hybridization, or molecular solubility. it can. For example, the deoxyribose phosphate backbone of nucleic acid molecules can be modified to yield peptide nucleic acids (Hyrup, B. and Nielsen, PE (1996) Bioorg. Med. Chem. 4 (1): 5-23). As used herein, the term “peptide nucleic acid” or “PNA” means a nucleic acid mimic, eg, a DNA mimic, that replaces the deoxyribose phosphate backbone with a pseudopeptide backbone and maintains only four natural nucleobases. . The natural backbone of PNA has been shown to allow DNA and RNA specific hybridization in a low ionic strength environment. The synthesis of PNA oligomers is carried out as described in standard Hyrup and Nielsen (1996) supra and Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93: 14670-675. Can be performed using the peptide method.

PNAs of B7-4 or PD-1 nucleic acid molecules can be used for therapeutic and diagnostic purposes. For example, PNA can be used as an antisense or antigenic agent for sequence-specific modulation of gene expression, for example by inducing transcription or translational arrest or inhibiting replication. Also, PNAs of B7-4 or PD-1 nucleic acid molecules can be used to analyze single base pair mutants in genes (eg, PNA-directed PCR restriction) when used in combination with other enzymes. As an “artificial restriction enzyme” (eg, the above S1 nuclease (Hyrup and Nielsen (1996) supra)) or as a DNA sequence or probe or primer for hybridization (Hyrup and Nielsen (1996) supra; Perry-O'Keefe et al. al. (1996) supra) can be used.
In other embodiments, the B7-4 or PD-1 PNA can be attached by attaching lipophilic or other helper groups to the PNA, by formation of a PNA-DNA chimera, or by liposomes or drug delivery known to those skilled in the art. Can be modified by other methods (eg, to increase stability or cell uptake). For example, PNA-DNA chimeras of B7-4 or PD-1 nucleic acid molecules can be obtained that can combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes (eg, RNAse H and DNA polymerase) to interact with the DNA portion, while the PNA portion provides high binding affinity and specificity. I will. PNA-DNA chimeras can be joined using linkers of appropriate lengths selected for base stacking, number of linkages between nucleobases, and orientation (Hyrup B. and Nielsen (1996) supra). Synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. and Nielsen (1996) supra and Finn PJ et al. (1996) Nucleic Acids Res. 24 (17): 3357-63. For example, DNA strands can be synthesized on a solid support using standard phosphoramide coupling chemistry. Modified nucleoside analogs (eg, 5 ′-(4-methoxytrityl) amino-5′-deoxy-thymidine phosphoramide) can be used as linkers between PNA and the 5 ′ end of DNA (Mag, M. et al. ( 1989) Nucleic acid Res. 17: 5973-88). The PNA monomer then couples with the 5 ′ PNA segment and the 3 ′ DNA segment in a stepwise manner to generate a chimeric molecule (Finn PJ et al. (1996) supra). Alternatively, chimeric molecules can be synthesized with a 5 ′ DNA segment and a 3 ′ PNA segment (Peterser, KH et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).

  In other embodiments, the oligonucleotides are other adducts such as peptides (eg, for targeting host cell receptors in vivo) or cell membranes (eg, Letsinger et al. (1989) Proc. Natl Acad. Sci. USA 86: 6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84: 648-652; Agents that promote penetration of WO 89/10134). In addition, oligonucleotides can be cleaved by hybridization (see, eg, Krol et al. (1988) Biotechniques 6: 958-976) or intercalating agents (see, eg, Zon (1988) Pharm. Res. 5: 539 -549). To this end, oligonucleotides can be conjugated to other molecules (eg, peptides, crosslinkers induced by hybridization, transport agents, or cleavage agents induced by hybridization).

III. Isolated B7-4 or PD-1 protein and anti-B7-4 or PD-1 antibody Further isolated B7-4 or PD-1 protein and biologically active portion thereof, and anti-B7-4 or PD -1 protein can also be used as a modulation agent. In one embodiment, native B7-4 or PD-1 protein can be isolated from cells or tissue sources by a suitable purification scheme using standard protein purification techniques. In one embodiment, the B7-4 or PD-1 protein is produced by recombinant DNA technology. As an alternative to recombinant expression, B7-4 or PD-1 protein or polypeptide can be chemically synthesized using standard peptide synthesis techniques.

  Another aspect of the invention relates to an isolated B7-4 or PD-1 protein. Preferably, the B7-4 or PD-1 protein comprises the amino acid sequence encoded by SEQ ID NO: 1, 3, 10, or 11. In another preferred embodiment, the protein comprises the amino acid sequence of SEQ ID NO: 2, 4, or 12. In other embodiments, the protein has at least 50%, at least 60% amino acid identity, more preferably 70% amino acid identity, more preferably 80%, and more preferably 90% or 95% amino acid identity. The amino acid sequence is shown in SEQ ID NO: 2, 4 or 12.

  In other embodiments, the present invention provides isolated portions of B7-4 or PD-1 protein. For example, the B7-4 protein includes a signal sequence and IgV and IgC domains. The signal sequence of SEQ ID NO: 2 is shown from around amino acid 1 to around amino acid 18. The signal sequence of SEQ ID NO: 4 is shown from around amino acid 1 to around amino acid 18. The IgV domain of SEQ ID NO: 2 is shown from about amino acid 19 to about amino acid 134, and the IgV domain of SEQ ID NO: 4 is shown from about amino acid 19 to about amino acid 134. The IgC domain of SEQ ID NO: 2 is shown from about amino acid 135 to about amino acid 227, and the IgC domain of SEQ ID NO: 4 is shown from about amino acid 135 to about amino acid 227. The hydrophilic tail of the B7-4 molecule exemplified in SEQ ID NO: 2 includes the hydrophilic tail shown from around amino acid 228 to around amino acid 245. The B7-4 polypeptide exemplified in SEQ ID NO: 4 comprises a transmembrane domain shown from about amino acid 239 to about amino acid 259 of SEQ ID NO: 4 and a cytoplasmic domain shown from about amino acid 260 to about amino acid 290 of SEQ ID NO: 4 including. PD-1 polypeptide is 288 amino acids long and its domain structure is known in the art (Shinohara et al. (1994) Genomics 23: 704). The predicted protein maturation form contains approximately 268 amino acids and includes an extracellular domain (147 amino acids), a transmembrane domain (27 amino acids), a transmembrane region (27 amino acids) and a cytoplasmic domain (94 amino acids). Four potential N-glycosylation sites are found in the extracellular domain (US Pat. No. 5,698,520). The 68 amino acid residues between the two cysteine residues (cys54 and cys123) have similarities to the disulfide-linked immunoglobulin domain of the V set sequence (US Pat. No. 5,698,520).

  The invention further relates to soluble forms of B7-4 or PD-1 protein. Such forms can be naturally occurring, eg, as shown in SEQ ID NO: 2, or can be genetically engineered and can include, for example, the extracellular domain of a B7-4 or PD-1 protein. A typical B7-4 extracellular domain comprises approximately amino acids 19-238 of SEQ ID NO: 4. A typical PD-1 extracellular domain comprises approximately amino acids 21-288 of SEQ ID NO: 12.

  In one aspect, the extracellular domain of the B7-4 polypeptide comprises mature forms of the B7-4 polypeptide, eg, IgV and IgC domains, but the transmembrane and cytoplasmic domains of the B7-4 polypeptide (eg, SEQ ID NO: 4 from amino acid 19 to amino acid 238) or SEQ ID NO: 2 from amino acid 19 to amino acid 245 is not included.

  In one aspect, the extracellular domain of the PD-1 polypeptide comprises a mature form of the PD-1 polypeptide, eg, an immunoglobulin superfamily domain (eg, a V set sequence), but the transmembrane domain of the PD-1 polypeptide. And the cytoplasmic domain (eg, around amino acids 21-288 of SEQ ID NO: 12) is not included.

  The biologically active portion of the B7-4 or PD-1 protein comprises a peptide that contains sufficient amino acid sequence from or derived from the amino acid sequence of the B7-4 or PD-1 protein It contains fewer amino acids than the long B7-4 or PD-1 protein and exhibits at least one activity of the B7-4 or PD-1 protein, preferably the ability to bind to a natural binding partner. Typically, the biologically active portion includes a domain or motif with at least one activity of a B7-4 or PD-1 protein. The biologically active portion of the B7-4 or PD-1 protein can be a polypeptide that is, for example, at least 10, 25, 50, 100, 150, 200 or more amino acids in length.

  To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for purposes of optimal comparison (eg, gaps are the first and second amino acids or A non-homologous sequence can be ignored for comparison purposes). In a preferred embodiment, the length of the reference sequence juxtaposed for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably the length of the reference sequence. Is at least 70%, 80% or 90%. The residues at the corresponding positions are then compared, and when a position in one sequence is occupied by the same residue as the corresponding position in another sequence, the molecules are identical at that position. Thus, the percent identity between two sequences is a function of the number of identical positions shared by the two sequences (ie,% identity = # of identical positions / total of positions # × 100). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, that need to be introduced for optimal alignment of the two sequences It is. As used herein, amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”.

  Sequence comparison and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is the brotham 62 matrix or PAM250 matrix, and the gap weight of 16, 14, 12, 10, 8, 6 or 4 and 1, 2, 3, 4 Measured using the GAP program in the GCG software package (available at http://www.gcg.com), using a length weight of 5 or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is NWSgapdna. CMP matrix and 40, 50, 60, 70 or 80 gap weight and 1, 2, 3, 4, 5 or 6 length weight. Using the GAP program in the GCG software package (available at http://www.gcg.com).

  In addition, by using the nucleic acid and protein sequences of the present invention as “query sequences”, searches against public databases can be performed, eg, other family members or related sequences can be identified. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215: 403-10. By performing a BLAST nucleotide search with the NBLAST program, score = 100, word length = 12, a nucleotide sequence homologous to the B7-4 or PD-1 nucleic acid molecule of the present invention is obtained. By performing the BLAST protein search with the XBLAST program, score = 50, word length = 3, an amino acid sequence homologous to the B7-4 or PD-1 protein molecule of the present invention can be obtained. Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25 (17): 3389-3402 to obtain a gapped alignment for comparative purposes. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used. For example, the nucleotide sequences of the present invention were analyzed using the default Blastn matrix 1-3 with gap penalties set at presence 11 and extension 1. Default settings: Amino acid sequences of the present invention were analyzed using a Brossum 62 matrix with gap penalties set at Existence 11 and Extension 1. See http://www.ncbi.nlm.nih.gov.

  The invention also provides a B7-4 or PD-1 chimera or fusion protein. As used herein, a B7-4 or PD-1 “chimeric protein” or “fusion protein” is a B7-4 or PD-1 operably linked to a non-B7-4 or PD-1 polypeptide. Including polypeptides. A “B7-4 or PD-1 polypeptide” includes a polypeptide having an amino acid sequence corresponding to a B7-4 or PD-1 polypeptide, and a “non-B7-4 or PD-1 polypeptide” includes a B7 -4 or a protein that is not substantially homologous to PD-1 protein, for example, a polypeptide having an amino acid sequence corresponding to a protein derived from the same or different organism, unlike B7-4 or PD-1 protein To do. Within the B7-4 or PD-1 fusion protein, the B7-4 or PD-1 polypeptide may correspond to all or part of the B7-4 or PD-1 protein. In a preferred embodiment, the B7-4 or PD-1 fusion protein comprises at least one biologically active portion of the B7-4 or PD-1 protein, such as the extracellular domain of the B7-4 or PD-1 protein. Within the fusion protein, the term “operably linked” means that the B7-4 or PD-1 polypeptide and the non-B7-4 or PD-1 polypeptide are fused in-frame to each other. It shall be shown that The non-B7-4 or PD-1 polypeptide can be fused to the N-terminus or C-terminus of the B7-4 or PD-1 polypeptide.

  For example, in one aspect, the fusion protein is a GST-B7-4 or GST-PD-1 fusion protein in which a B7-4 or PD-1 sequence is fused to the C-terminus of the GST sequence. In another embodiment, the fusion protein is B7-4 in which the B7-4 or PD-1 nucleotide sequence is inserted into a vector, such as a pCEP4-HA vector (Herrscher, RF et al. (1995) Genes Dev. 9: 3067-3082). 4 or PD-1-HA fusion protein, for example, the B7-4 or PD1 sequence is fused in-frame to an influenza hemagglutinin epitope tag. Such a fusion protein can facilitate the purification of recombinant B7-4 or PD-1 protein.

  B7-4 or PD-1 fusion protein corresponds to a nucleotide sequence encoding a first peptide having B74 activity and a moiety that alters the solubility, affinity, stability or valency of the first peptide, eg, an immunoglobulin constant region Can be produced by recombinant expression of a nucleotide sequence encoding the second peptide. Preferably, the first peptide is a portion of a B7-4 polypeptide (eg, amino acid residues 1- 1 of the sequence shown in SEQ ID NO: 4 that is sufficient to modulate costimulation or inhibition of activated immune cells). 238 or 19-238 (after signal sequence cleavage). In another preferred embodiment, the first peptide comprises a portion of the PD-1 polypeptide (eg, an amino acid residue of the sequence shown in SEQ ID NO: 12 that is sufficient to modulate costimulation or inhibition of activated immune cells). Consists of part 1-288 (or part of 21-288 after signal peptide cleavage). The second peptide may be an immunoglobulin constant region, such as human Cγ1 domain or Cγ4 (eg, human IgCγ1 or human IgCγ4 hinge, CH2 and CH3 regions, see eg Capon et al., US Pat. As part of the specification). The resulting fusion protein may have modified B7-4 or PD-1 solubility, binding affinity, stability and / or valency (ie, number of binding sites available per molecule) and protein purification Can increase efficiency. Fusion proteins and peptides produced by recombinant techniques can be secreted and isolated from a mixture of cells and media containing the protein or peptide. Alternatively, the protein or peptide can be retained by the cytoplasm, the cells harvested, lysed and the protein isolated. A cell culture typically includes host cells, media and other byproducts. Suitable media for cell culture are known in the art. Proteins and peptides can be isolated from cell culture media, host cells, or both using techniques known in the art for protein and peptide purification. Techniques for transfecting host cells and purifying proteins and peptides are known in the art.

  Particularly preferred B7-4 or PD-1 Ig fusion proteins comprise the extracellular domain portion or variable region-like domain of human B7-4 or PD-1 bound to an immunoglobulin constant region (eg, Fc region). The immunoglobulin constant region can include genetic modifications that reduce or eliminate effector activity inherent in immunoglobulin structures. For example, DNA encoding the extracellular portion of a B7-4 or PD-1 polypeptide may be a human IgGγ1 and / or IgGγ4 hinge modified by site-directed mutagenesis, for example as shown in WO 97/28267. , Linked to DNA encoding the CH2 and CH3 regions.

  Preferably, the B7-4 or PD-1 fusion protein of the present invention is produced by standard recombinant DNA techniques. For example, DNA fragments encoding different polypeptide sequences are ligated together in frame according to conventional techniques, for example, ligated using blunt or staggered ends and provided with suitable ends by restriction enzyme digestion. If appropriate, the cohesive ends are replenished, and alkaline phosphatase treatment avoids unwanted ligation and causes the enzyme ligation reaction. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers, which produce a complementary overhang between two consecutive gene fragments, followed by annealing and re-amplification to produce a chimeric gene sequence. (See, eg, Current Protocols in Molecular Biology, edited by Ausubel et al., John Willie and Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (eg, a GST polypeptide or HA epitope tag). A nucleic acid encoding B7-4 or PD-1 can be cloned into such an expression vector such that the fusion moiety is fused in-frame to the B7-4 or PD-1 protein.

  In another aspect, the fusion protein is a B7-4 or PD-1 protein containing a heterologous signal sequence at its N-terminus. In certain host cells (eg, mammalian host cells), expression and / or secretion of B7-4 or PD-1 can be enhanced through the use of heterologous signal sequences.

  The B7-4 or PD-1 fusion protein of the invention can be incorporated into a pharmaceutical composition and administered in vivo to a subject. The use of B7-4 or PD-1 fusion proteins is therapeutically useful in the treatment of immune diseases, such as autoimmune diseases, or transplant rejection inhibition cases. Furthermore, by using the B7-4 or PD-1 fusion protein of the present invention as an immunogen, an anti-B7-4 or PD-1 antibody is produced in the subject, and B7-4 or PD-1 is purified and screened. Assays can identify molecules that block the interaction of B7-4 with B7-4 receptors, such as PD-1.

  Preferably, the B7-4 or PD-1 chimera or fusion protein of the present invention is produced by standard recombinant DNA techniques. For example, DNA fragments encoding different polypeptide sequences are ligated together in frame according to conventional techniques, for example, ligated using blunt or staggered ends and provided with suitable ends by restriction enzyme digestion. If appropriate, the cohesive ends are replenished, and alkaline phosphatase treatment avoids unwanted ligation and causes the enzyme ligation reaction. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers, which produce a complementary overhang between two consecutive gene fragments, followed by annealing and re-amplification to produce a chimeric gene sequence. (See, eg, Current Protocols in Molecular Biology, edited by Ausubel et al., John Willie and Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (eg, a GST polypeptide). A nucleic acid encoding B7-4 or PD-1 can be cloned into such an expression vector such that the fusion moiety is fused in-frame to the B7-4 or PD-1 protein.

  The invention also relates to variants of the B7-4 or PD-1 protein that function as B7-4 or PD-1 agonists (mimetics) or as B7-4 or PD-1 antagonists. Variants of B7-4 or PD-1 protein can be generated by mutagenesis, eg, discrete point mutations or truncations in the B7-4 or PD-1 protein. An agonist of a B7-4 or PD-1 protein may retain substantially the same or a subset of the biological activity of the naturally occurring form of the B7-4 or PD-1 protein. An antagonist of the B7-4 or PD-1 protein may be used to inhibit the activity of the naturally occurring form of the B7-4 or PD-1 protein, eg, by competitively modulating the cellular activity of the B7-4 or PD-1 protein. One or more may be inhibited. That is, specific biological effects can be induced by treatment with restricted function variants. In one aspect, treating a subject with a variant having a biologically active subset of the naturally occurring form of the protein has fewer side effects in the subject than when treated with the naturally occurring form of the B7-4 or PD-1 protein. It was.

  In one embodiment, a variant of a B7-4 or PD-1 protein that functions as a B7-4 or PD-1 agonist (mimetics) or as a B7-4 or PD-1 antagonist is a B7-4 or PD-1 protein agonist. Alternatively, it can be identified by screening a combinatorial library of mutants of the B7-4 or PD-1 protein, such as a truncated fragment for antagonist activity. In one embodiment, a miscellaneous library of B7-4 or PD-1 variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a miscellaneous gene library. A variegated library of B7-4 or PD-1 variants can be produced, for example, by enzymatically linking a mixture of synthetic oligonucleotides to a gene sequence, in which case the potential B7-4 or PD A degenerate set of -1 sequences can be expressed as individual polypeptides or alternatively as a set of large fusion proteins (eg, in the case of phage display) containing a set of B7-4 or PD-1 sequences therein It is. There are various methods that can be used to produce a library of potential B7-4 or PD-1 variants from a degenerate oligonucleotide sequence. Chemical synthesis of degenerate gene sequences can be performed on an automated DNA synthesizer, and the synthetic gene can then be ligated into an appropriate expression vector. The use of a degenerate set of genes can provide all the sequences that encode the desired set of potential B7-4 or PD-1 sequences in a mixture. Methods for synthesizing degenerate oligonucleotides are known in the art (eg, Narang, SA (1983) Tetrahedron 39: 3, Itakura et al. (1984) Annu. Rev. Biochem. 53: 323, Itakura et al. (1984) Science 198: 1056, Ike et al. (1983) Nucleic Acid Res. 11: 477).

  In addition, a library of B7-4 or PD-1 protein coding sequence fragments is used to generate a miscellaneous population of B7-4 or PD-1 fragments to produce variants of the B7-4 or PD-1 protein. Can be screened and subsequently selected. In one embodiment, the library of coding sequence fragments may contain sense / antisense pairs from different nick products by nicking approximately once per molecule, denaturing double-stranded DNA and regenerating the DNA. Under conditions that allow double-stranded DNA to form, remove the single-stranded portion from the reshaped duplex by S1 nuclease treatment, and bind the resulting fragment library to an expression vector, B7-4 or PD-1 It can be generated by processing a double stranded PCR fragment of the coding sequence. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of B7-4 or PD-1 protein.

  Several techniques are known in the art for screening gene products in combinatorial libraries produced by point mutation or truncation, and for screening cDNA libraries for gene products with selected properties. Yes. The above technique can be adapted for rapid screening of gene libraries generated by combinatorial mutagenesis of B7-4 or PD-1 proteins. The most widely used technique for screening large gene libraries with high throughput analysis is typically the cloning of gene libraries into replicable expression vectors and the appropriate library of generated vectors. Transforming the cells and expressing the combined gene under conditions that facilitate detection of the desired activity and isolation of the vector encoding the gene from which the product was detected. By using a new technique that increases the frequency of functional mutants in libraries, called Recursive ensemble mutagenesis (REM), in combination with screening assays, B7-4 or PD-1 variants Can be identified (Arkin and Youvan (1992) Proc. Natl. Acad. Sci. USA 89: 7811-7815, Delagrave et al. (1993) Protein Eng. 6 (3): 327-331).

  In one aspect, miscellaneous B7-4 or PD-1 libraries can be analyzed by utilizing cell-based assays. For example, a library of expression vectors can be transfected into cell lines that normally synthesize and secrete B7-4 or PD-1. The transfected cells are then cultured such that B7-4 or PD-1 and the specific mutant B7-4 or PD-1 are secreted, and the mutation to B7-4 or PD-1 activity in the cell supernatant Body expression effects can be detected, for example, by any of a number of functional assays. Plasmid DNA is then recovered from the cells and evaluated for inhibition or alternatively enhancement of B7-4 or PD-1 activity, and individual clones can be further characterized.

  In addition to B7-4 or PD-1 polypeptides composed only of naturally occurring amino acids, B7-4 or PD-1 peptide mimetics are also provided. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties similar to those of template peptides. These types of non-peptide compounds are referred to as “peptide mimetics” or “peptide mimetics” (Fauchere, J. (1986) Adv. Drug Res. 15:29, Veber and Freidinger (1985) TINS 392, And Evans et al. (1987) J. Med. Chem. 30: 1229, incorporated herein by reference), usually developed with the aid of computerized molecular modeling. By using peptide mimetics that are structurally similar to therapeutically useful peptides, an equivalent content of therapeutic or prophylactic effect can be produced. In general, peptidomimetics are structurally similar to paradigm polypeptides (ie, polypeptides having biological or pharmacological activity) such as human B7-4 or PD-1, but are known in the art. In addition, in the following references: Spatola, AF, “Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins”, edited by Weinstein, B., Marcel Dekker, New York, 267 (1983), Spatola, AF, Vega Data (1983). March 1), Volume 3, 3rd Edition, “Peptide Backbone Modifications” (Overview), Morley, JS (1980) Trends Pharm. Sci. 463-468 (Overview), Hudson, D. et al. (1979) Int. J. Pept. Prot. Res. 14: 177-185 (-CH2NH, CH2CH2-), Spatola, AF et al. (1986) Life Sci. 38: 1243-1249 (-CH2-S), Hann, MM (1982) J .Chem.Soc.Perkin Trans.I.307 314 (-CH-CH-, cis and trans), Almquist, RG et al. (190) J. Med. Chem. 23: 1392-1398 (-COCH2-), Jennings-White, C. et al. (1982) Tetrahedron Lett. 23: 2533 (-COCH2-), Szelke, M. et al., European application EP 45665 (1982) CA: 97: 39405 (1982) (-CH (OH) CH2-), Holladay, MW et al. (1983) Tetrahedron Lett. 1983) 24: 4401-4404 (-C (OH) CH2-), and Hruby, VJ (1982) Life Sci. (1982) 31: 189-199 (-CH2-S-) ( Each of which is incorporated herein by reference), -CH2NH-, CH2S-, -CH2-CH2-, -CH = CH- (cis and trans), -COCH2-, -CH (OH ) Having one or more peptide bonds optionally substituted by a bond selected from the group consisting of CH2- and -CH2SO-. A particularly preferred non-peptide bond is —CH 2 NH—. Such peptidomimetics are, for example, more economical in productivity, high chemical stability, high pharmacological properties (half-life, absorption, efficacy, potency, etc.), modified in specificity It may have significant advantages over polypeptide embodiments, including (eg, broad spectrum biological activity), low antigenicity, and the like. Peptide mimetic labeling, either directly or via a spacer (eg, an amide group), to non-interfering position (s) on the peptide mimetic predicted by quantitative structure activity data and / or molecular modeling Usually accompanied by covalent attachment of one or more of the labels. The non-interfering position is a position that generally does not form direct contact with the macromolecule (s) that produce a therapeutic effect upon binding of peptide mimetics. Peptidomimetics derivatization (eg, labeling) should not substantially interfere with the intended biological or pharmacological activity of the peptide mimetic.

  By using a method of systematically replacing one or more amino acids of the B7-4 or PD-1 amino acid sequence with a D-amino acid of the same type (eg, D-lysine instead of L-lysine), Stable peptides can be produced. In addition, constrained peptides containing B7-4 or PD-1 amino acid sequences or substantially identical sequence changes add, for example, internal cysteine residues that can form intramolecular disulfide bridges that ring the peptide. Can be produced by methods known in the art (Rizo and Gierasch (1992) Annu. Rev. Biochem. 61: 387, hereby incorporated by reference).

  According to the amino acid sequence of the B7-4 or PD-1 polypeptide identified here, one skilled in the art would be able to produce a polypeptide corresponding to the B7-4 or PD-1 peptide sequence and its sequence variants. Should be possible. Such polypeptides can be produced in prokaryotic or eukaryotic host cells by expression of a polynucleotide encoding a B7-4 or PD-1 peptide sequence, often as part of a larger polypeptide. Alternatively, the peptide can be synthesized by chemical methods. Methods for the expression of heterologous proteins in recombinant hosts, chemical synthesis of polypeptides and in vitro translation are well known in the art and are described in Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd edition, Cold Spring Harbor. New York, Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Incorporated, San Diego, California, Merrifield, J. (1969) J. Am. Chem. Soc. 91. : 501, Chaiken IM (1981) CRC Crit. Rev. Biochem. 11: 255, Kaiser et al. (1989) Science 243: 187, Merrifield, B. (1986) Science 232: 342, Kent, SBH (1988) Annu. Rev. Biochem. 57: 957, and Offord, RE (1980) Semisynthetic Proteins, Willy Publishing (these Are hereby incorporated by reference).

  Peptides are typically produced by direct chemical synthesis and can be used, for example, as agonists or antagonists of the B7-4 / PD-1 interaction. Peptides can be produced as modified peptides in which non-peptide moieties are linked by covalent bonds to the N-terminus and / or C-terminus. In certain preferred embodiments, the carboxy terminus or amino terminus, or both, are chemically modified. The most common modifications of the terminal amino and carboxy groups are acetylation and amidation, respectively. Amino terminal modifications, such as acylation (eg, acetylation) or alkylation (eg, methylation) and carboxy terminal modifications, such as amidation, and other terminal modifications, such as ring closure, are incorporated into various aspects of the invention. obtain. Certain amino- and / or carboxy-terminal modifications and / or peptide extensions to the core sequence enable advantageous physical, chemical, biochemical and pharmacological properties such as high stability, strong potency and / or efficacy Sex, resistance to serum proteases, desirable pharmacokinetic properties, etc. may be provided. Diseases can be treated by using peptides for therapy, for example by altering costimulation in a patient.

  Using an isolated B7-4 or PD-1 protein, or a portion or fragment thereof (or a nucleic acid molecule encoding such a polypeptide) as an immunogen, by using standard polyclonal and monoclonal antibody production techniques. Antibodies that bind to B7-4 or PD-1 can be produced. Full length B7-4 or PD-1 protein can also be used, or alternatively, the present invention provides an antigenic peptide fragment of B7-4 or PD-1 for use as an immunogen. Since the antigenic peptide of B7-4 or PD-1 contains at least 8 amino acid residues and contains an epitope of B7-4 or PD-1, the antibody produced against the peptide is B7-4 or PD A specific immune complex with -1. Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues. Including.

  Alternatively, antigenic peptide fragments of B7-4 or PD-1 polypeptide can be used as an immunogen. An antigenic peptide fragment of a B7-4 or PD-1 polypeptide typically comprises at least 8 amino acid residues of the amino acid sequence set forth in SEQ ID NO: 2, 4 or 12, and B7-4 or PD- By including an epitope of one polypeptide, antibodies raised against the peptide form an immune complex with the B7-4 or PD-1 molecule. A preferred epitope contained in the antigenic peptide is a B7-4 or PD-1 region located on the protein surface, such as a hydrophilic region. In one embodiment, the antibody binds substantially specifically to a B7-4 or PD-1 molecule. In another embodiment, the antibody specifically binds to a B7-4 or PD-1 polypeptide.

  Preferably, the antigenic peptide has at least about 10 amino acid residues, more preferably at least about 15 amino acid residues, more preferably at least about 20 amino acid residues, and most preferably at least about 30 amino acid residues. Contains amino acid residues. A preferred epitope contained in an antigenic peptide is a region of B7-4 or PD-1 polypeptide located on the protein surface, such as a hydrophilic region, and is unique to B7-4 or PD-1 polypeptide. In one embodiment, the epitope may be specific for a B7-4 or PD-1 protein from one species, such as mouse or human (ie, B7-4 or PD that is not conserved across species). Antigenic peptides spanning the region of -1 polypeptide are used as immunogens, the non-conserved residues can be measured using alignments such as those described herein). By performing a standard hydrophobicity analysis of the B7-4 or PD-1 protein, the hydrophilic region can be identified.

  Antibodies are produced by immunizing a suitable subject (eg, rabbit, goat, mouse or other mammal) with the immunogen, typically using B7-4 or PD-1 immunogen. Suitable immunogenic products may include, for example, recombinantly expressed B7-4 or PD-1 protein or chemically synthesized B7-4 or PD-1 peptide. In addition, the product may contain an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of an appropriate subject with an immunogenic B7-4 or PD-1 product induces a polyclonal anti-B7-4 or PD-1 antibody response.

  In another embodiment, the nucleic acid vaccine is a DNA-coated gold particle on the epidermis by various means such as injection (eg, intramuscular, intradermal, or gene accelerator that injects the particles into the skin using a particle accelerator or compressed gas) (Haynes et al., 1996, J. Biotechnol. 44:37). Alternatively, the nucleic acid vaccine can be administered by non-invasive means. For example, pure or lipid formulated DNA can be delivered to the respiratory system or other targeted locations, eg, Peyer's patches by oral delivery of DNA (Schubbert, 1997, Proc. Natl. Acad. Sci. USA 94 : 961). Attenuated microorganisms can be used for delivery to mucosal surfaces (Sizemore et al., 1995, Science, 270: 29).

  Polyclonal anti-B7-4 or PD-1 antibodies can be produced as described above by immunizing a suitable subject with a B7-4 or PD-1 immunogen. Anti-B7-4 or PD-1 antibody titer in the immunized subject is monitored over time by standard techniques, such as an enzyme linked immunosorbent assay (ELISA) using immobilized B7-4 or PD-1 polypeptide. obtain. If desired, antibody molecules directed against B7-4 or PD-1 polypeptides can be isolated from mammals (eg, from blood) and further purified by known techniques, such as protein A chromatography, to yield IgG fractions. It is done. At an appropriate time after immunization, for example when anti-B7-4 or PD-1 antibody titers are highest, antibody producing cells are obtained from the subject and standard techniques such as Kohler and Milstein (1975) Nature 256: 495. -497, also described by Brown et al. (1981) J. Immunol. 127: 539-46, Brown et al. (1980) J. Biol. Chem. 255: 4980-83, Yeh et al. (1976). Proc. Natl. Acad. Sci. 76: 2927-31 and Yeh et al. (1982) Int. J. Cancer 29: 269-75), more recent human B cell hybridoma technology (Kozbor et al. (1983) Immunol. Today 4:72), by EBV-hybridoma technology (Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Lis, Inc., pp. 77-96) or by trioma technology It can be used in the production of monoclonal antibodies. Techniques for producing monoclonal antibody hybridomas are known (generally, Kenneth, RH, Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corporation, New York, New York (1980), Lerner, EA (1981) Yale J Biol. Med. 54: 387-402, Gefter, ML et al. (1977) Somatic Cell Genet. 3: 231-36). Briefly, immortal cell lines (typically myeloid species) are fused to lymphocytes (typically spleen cells) from mammals immunized with B7-4 or PD-1 immunogen as described above. Upon screening the culture supernatant of the generated hybridoma cells, hybridomas producing monoclonal antibodies that preferably bind specifically to the B7-4 or PD-1 polypeptide are identified.

  Any of a number of known protocols used for fusion of lymphocytes and immortalized cell lines can be applied for anti-B7-4 or PD-1 monoclonal antibody production purposes (eg, Galfre, G. et al. (1977) Nature 266). : 55052, Gefter et al. (1977) supra, Lerner (1981) supra, Kenneth (1980) supra). Furthermore, it will be apparent to those skilled in the art that there are many variations of the above method that are also useful. Typically, immortal cell lines (eg, myeloma cell lines) are derived from the same species of mammal as the lymphocytes. For example, murine hybridomas can be produced by fusing lymphocytes from mice immunized with an immunogenic product of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a number of myeloma cell lines can be used as fusion partners by standard techniques, such as P3-NS1 / 1-Ag4-1, P3-x63-Ag8.653 or Sp2 / O-Ag14 myeloma lines. is there. These myeloid lineages are available from the American Type Culture Collection (ATCC) in Rockville, Maryland. Typically, HAT-sensitive mouse myeloma cells are fused to mouse spleen cells using polyethylene glycol (“PEG”). Hybridoma cells generated from the fusion are then selected using HAT medium, in which non-fused and non-productive fused myeloma cells are killed (unfused spleen cells die after a few days because they are not transformed). . Hybridoma cells producing the monoclonal antibodies of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind to B7-4 or PD-1 molecules using, for example, standard ELISA assays.

  As another method for producing a monoclonal antibody-secreting hybridoma, a monoclonal anti-B7-4 or PD-1 antibody is screened for a recombinant combinatorial immunoglobulin library (eg, antibody phage display library) with B7-4 or PD-1. Can be identified and isolated by isolating immunoglobulin library members that bind to the B7-4 or PD-1 polypeptide. Kits for creating and screening phage display libraries are commercially available (eg, Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01, and Stratagene SurfZAP ™ Phage Display Kit). Catalog number 240612). In addition, examples of methods and reagents that are particularly amenable to use in generating and screening antibody display libraries include, for example, Ladner et al., US Pat. No. 5,223,409, Kang et al., International Publication No. WO 92/18619, Dower et al., International Publication No. WO 91 / 17271, Winter et al., International Publication No. WO 92/20791, Markland et al., International Publication No. WO 92/15679, Breitling et al., International Publication WO 93/01288, McCafferty et al., International Publication No. WO 92/01047, Garrard et al., International Publication No. WO 92/09690, Ladner et al., International Publication No. WO 90/02809, Fuchs et al. (1991) Biotechnology (NY) 9: 1369-1372, Hay et al. (1992) Hum. Antibod. Hybridomas 3: 81-85, Huse et al. (1989) Science 246: 1275- 1281, Griffiths et al. (1993) EMBO J. 12: 7 5-734, Hawkins et al. (1992) J. Mol. Biol. 226: 889-896, Clarkson et al. (1990) Nature 352: 624-628, Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89: 3576-3580. Garrard et al. (1991) Biotechnology (NY) 9: 1373-1377, Hoogenboom et al. (1991) Nucleic Acids Res. 19: 4133-4137, Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88: 7978-7882. And McCafferty et al. (1990) Nature 348: 552-554.

  In addition, recombinant anti-B7-4 or PD-1 antibodies, such as chimeric and humanized monoclonal antibodies, include both human and non-human portions and can be produced using standard recombinant DNA techniques. Included within the scope of the invention. Such chimeric and humanized monoclonal antibodies are described, for example, in Robinson et al., International Patent Publication PCT / US86 / 02269, Akira et al., European Patent Application 184187, Taniguchi, M., European Patent Application 17196, Morrison et al., European Patent Application 173494, Neuberger et al. PCT application WO 86/01533, Cabilly et al., US Pat. No. 4,816,567, Cabilly et al., European patent application 125023, Better et al. (1988) Science 240: 1041-1043, Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84: 3439-3443, Liu et al. (1987) J. Immunol. 139: 3521-3526, Sun et al. (1987) Proc. Natl. Acad. Sci. 84: 214-218, Nishimura et al. (1987) Cancer Res. 47: 999-. 1005, Wood et al. (1985) Nature 314: 446-449, and Shaw et al. (19 8) J. Natl. Cancer Inst. 80: 1553-1559, Morrison, SL (1985) Science 229: 1202-1207, Oi et al. (1986) Biotechniques 4: 214, Winter US Pat. No. 5,225,539, Jones et al. (1986) Nature 321: 552. -525, Verhoeyan et al. (1988) Science 239: 1534, and Beidler et al. (1988) J. Immunol. 141: 4053-4060 can be produced by recombinant DNA techniques known in the art. .

  In addition, humanized antibodies can be produced according to standard protocols, such as those disclosed in US Pat. No. 5,565,332. In another embodiment, the antibody chain or specific binding pair component can be replicated using techniques known in the art, eg, as described in US Pat. No. 5,565,332, 5871907 or 5733743, and the specific binding pair component polypeptide chain and replicable. It can be produced by recombination between a vector containing a nucleic acid molecule encoding a one-component fusion of a gene display package and a vector containing a nucleic acid molecule encoding a second polypeptide chain of a single bond pair component. Inhibition of protein function in cells by the use of intracellular antibodies is also known in the art (eg Carlson, JR (1988) Mol. Cell. Biol. 8: 2638-2646, Biocca, S. et al. (1990)). EMBO J. 9: 101-108, Werge, TM et al. (1990) FEBS Lett. 274: 193-198, Carlson, JR (1993) Proc. Natl. Acad. Sci. USA 90: 7427-7428, Marasco, WA et al. (1993) Proc. Natl. Acad. Sci. USA 90: 7889-7893, Biocca, S. et al. (1994) Biotechnology (NY) 12: 396-399, Chen, SY et al. (1994) Hum. Gene Ther. 5: 595. -601, Duan, L. et al. (1994) Proc. Natl. Acad. Sci. USA 91: 5075-5079, Chen, SY et al. (1994) Proc. Natl. Acad. Sci. USA 91: 5932-5936, Beerli, RR et al. (1994) J. Biol. Chem. 269: 23931-23936, Beerli, RR et al. (1994) Biochem. Bio. Phys. Res. Commun. 204: 666-672, Mhashilkar, AM et al. (1995) EMBO J. 14: 1542-1551, Richardson, JH et al. (1995) Proc. Natl. Acad. Sci. USA 92: 3137-3141, Marasco PCT publication number WO 94/02610 by Du et al. And PCT publication number WO 95/03832 by Duan et al.).

  In one embodiment, the antibody used in the present invention is a bispecific antibody. Bispecific antibodies have binding sites for two different antigens within a single antibody molecule. Antigen binding can be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Examples of bispecific antibodies produced by hybrid hybridomas or triomas are disclosed in US Pat. No. 4,474,893. Bispecific antibodies can be synthesized by chemical means (Staerz et al. (1985) Nature 314: 628, and Perez et al. (1985) Nature 316: 354) and hybridoma technology (Staerz and Bevan (1986) Proc. Natl. Acad. Sci. USA, 83: 1453, and Staerz and Bevan (1986) Immunol, Today 7: 241). Bispecific antibodies are also described in US Pat. No. 5,959,084. Bispecific antibody fragments are described in US Pat. No. 5,798,229.

  Bispecific agents are also produced by identifying clones that produce heterologous hybridomas by fusing hybridomas or other cells that produce different antibodies, then produce and co-assembling both antibodies. Can be done. They can also be produced by chemical or genetic conjugation of complete immunoglobulin chains or portions thereof, such as Fab and Fv sequences. The antibody component can bind to PD-1 or B7-4.

Using anti-B7-4 or PD-1 antibodies (eg, monoclonal antibodies), B7-4 or PD-1 polypeptides can be isolated by standard techniques such as affinity chromatography or immunoprecipitation. Purification of native B7-4 or PD-1 polypeptide from cells and genetically recombinantly produced B7-4 or PD-1 polypeptide expressed in cells by anti-B7-4 or PD-1 antibodies Can be easy. Furthermore, anti-B7-4 or PD-1 antibodies can be used for detection of B7-4 or PD-1 protein (eg, in cell lysates or cell supernatants). Detection can be facilitated by coupling (ie, physically linking) the antibody to a detectable substance. Thus, in one embodiment, the anti-B7-4 or PD-1 antibody of the invention is labeled with a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent (luminescent) materials and radioactive materials. Examples of suitable enzymes are horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase, examples of suitable prosthetic group complexes are streptavidin / biotin and avidin / biotin, suitable fluorescence Examples of substances are umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin, examples of luminescent substances are luminol, and suitable radioactive substances Examples are 125 I, 131 I, 35 S and 3 H.

Yet another aspect of the invention relates to an anti-B7-4 or PD-1 antibody obtained by a method comprising the following steps:
(A) immunizing an animal with an immunogenic B7-4 or PD-1 protein or an immunogenic portion thereof specific to a B7-4 or PD-1 polypeptide, and (b) a B7-4 or PD-1 protein Antibodies that specifically bind to are isolated from the animal.

IV. Recombinant Expression Vectors and Host Cells Nucleic acid molecules encoding B7-4 or PD-1 family proteins (or portions thereof) can be included in vectors, preferably expression vectors. As used herein, the term “vector” includes a nucleic acid molecule capable of transporting another nucleic acid bound thereto. One type of vector is a “plasmid”, which includes a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors may direct the expression of genes that are operably linked to them. Such vectors are referred to herein as “expression vectors”. In general, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the present invention is considered to include such other forms of expression vectors that perform equivalent functions, such as viral vectors (eg, replication deficient retroviruses, adenoviruses and adeno-associated viruses).

  A recombinant expression vector may comprise a nucleic acid molecule of the invention in a form suitable for expression, eg, constitutive or inducible expression, of a PD-1 or B7-4 molecule in the indicator cell (s) of the nucleic acid in a host cell. This means that the recombinant expression vector contains one or more regulatory sequences selected based on the host cell used for expression and functions on the nucleic acid sequence to be expressed. Combined to get. Within a recombinant expression vector, “operably linked” means that the nucleotide sequence of interest is of a nucleotide sequence (eg, in an in vitro transcription / translation system or the vector is introduced into a host cell). It is meant to be linked to regulatory sequence (s) in a state that allows expression in the host cell. The term “regulatory sequence” includes promoters, enhancers and other expression control elements (eg, polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel (1990) Methods Enzymol. 185: 3-7. Regulatory sequences include those that direct constitutive expression of nucleotide sequences in many types of host cells and those that direct expression of nucleotide sequences only in certain host cells (eg, tissue-specific regulatory sequences). One skilled in the art will recognize that the design of an expression vector can vary depending on factors such as the choice of host cell to be transformed and the level of expression of the target protein. The expression vector of the present invention can be introduced into a host cell, as described herein (eg, B7-4 or PD-1 family protein, B7-4 or PD-1 protein mutant form, A protein or peptide comprising a fusion protein or peptide encoded by a nucleic acid may be produced.

  The recombinant expression vectors of the invention can be designed for the expression of B7-4 or PD-1 protein in prokaryotic or eukaryotic cells. For example, the B7-4 or PD-1 protein can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are further discussed in Goeddel (1990) supra. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

  Protein expression in prokaryotes is often performed in E. coli with a vector containing a constitutive or inducible promoter that directs the expression of a fused or non-fused protein. A fusion vector adds some amino acids to the protein encoded therein, usually at the amino terminus of the recombinant protein. Such fusion vectors are typically useful in the purification of recombinant proteins by 1) increasing the expression of the recombinant protein, 2) increasing the solubility of the recombinant protein, and 3) acting as a ligand in affinity purification. It is suitable for three purposes. In many cases, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein so that the recombinant protein can be separated from the fusion moiety following purification of the fusion protein. Among the enzymes and their cognate recognition sequences are factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc., Smith, DB and Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) And pRIT5 (Pharmacia, Piscataway, New Jersey), each of which fuses glutathione S-transferase (GST), maltose E binding protein or protein A to the target recombinant protein.

  Purified fusion proteins can be used in B7-4 or PD-1 activity assays (eg, direct assays or competitive assays detailed below) or to generate antibodies specific for, eg, B7-4 or PD-1 proteins. Can be used.

  Examples of suitable inducible unfused E. coli expression vectors include pTrc (Amann et al. (1988) Gene 69: 301-315) and pET11d (Studier et al. (1990) Methods Enzymol. 185: 60-89. ) Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET11d vector relies on transcription from a T7gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7gn1). This viral polymerase is supplied by host strains BL21 (DE3) or HMS174 (DE3) from an endogenous prophage harboring a T7gn1 gene under the transcriptional control of the lacUV5 promoter.

  One strategy to maximize recombinant protein expression in E. coli is to express the protein in host bacteria that have impaired ability to proteolytically cleave the recombinant protein (Gottesman, S. (1990) Methods Enzymol. 185: 119-128). Another strategy is to modify the nucleic acid sequence of the nucleic acid that is inserted into the expression vector so that the individual codons for each amino acid are preferentially utilized in E. coli. (Wada et al. (1992) Nucleic Acids Res. 20: 2111-2118). Such modifications of the nucleic acid sequences of the invention can be performed by standard DNA synthesis techniques.

  In another embodiment, the B7-4 or PD-1 expression vector is a yeast expression vector. Examples of vectors expressed in the yeast S. cerevisiae include pYepSec1 (Baldari et al. (1987) EMBO J. 6: 229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943), pJRY88 (Schultz et al. (1987) Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, California) and picZ (Invitrogen Corporation, San Diego, California).

  Alternatively, B7-4 or PD-1 polypeptide can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for protein expression in cultured insect cells (eg, Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklow, VA and Summers). MD (1989) Virology 170: 31-39).

  In yet another aspect, the nucleic acids of the invention are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pMex-NeoI, pCDM8 (Seed, B. (1987) Nature 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells, see Sambrook, J. et al., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, See chapters 16 and 17 of Cold Spring Harbor, New York (1989).

  In another embodiment, the recombinant mammalian expression vector can preferentially direct the expression of a nucleic acid in a particular cell type (eg, tissue-specific regulatory elements are used for expression of the nucleic acid). Tissue specific regulatory elements are known in the art. Non-limiting examples of suitable tissue specific promoters include albumin promoters (liver specific, Pinkert et al. (1987) Genes Dev. 1: 268-277), lymph specific promoters (Calame and Eaton (1988) Adv. Immunol. 43: 235-275), particularly T cell receptor promoters (Winoto and Baltimore (1989) EMBO J. 8: 729-733) and immunoglobulins (Banerji et al. (1983) Cell 33: 729-740, Queen and Baltimore ( 1983) Cell 33: 741-748), neuron specific promoters (eg, neurofilament promoters, Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas specific promoters (Edlund et al. (1985) ) Science 230: 912-916), and mammary gland specific promoters (eg, whey) Promoters, U.S. Pat. No. 4,873,316 and European Application No. 264166). Also included are developmentally regulated promoters, such as the murine hox promoter (Kessel and Gruss (1990) Science 249: 374-379) and the alpha-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3: 537-546). Is done.

  In addition, inducible regulatory systems used in mammalian cells are known in the art, for example, gene expression is heavy metal ions (eg, Mayo et al. (1982) Cell 29: 99-108, Brinster et al. (1982) Nature 296: 39- 42, Seelele et al. (1985) Mol. Cell. Biol. 5: 1480-1489), heat shock (eg, Nouer et al. (1991) Heat Shock Response, Nouer, L., CRC, Boca Raton, Florida, 167- 220), hormones (eg, Lee et al. (1981) Nature 294: 228-232, Hynes et al. (1981) Proc. Natl. Acad. Sci. USA 78: 2038-2042, Klock et al. (1987) Nature 329: 734-736. , Israel and Kaufman (1989) Nucl. Acids Res. 17: 2589-2604, and PCT Publication No. WO 93/23431), FK506-related molecules (eg, PCT publication). No. WO 94/18317) or tetracyclines (Gossen, M. and Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89: 5547-5551, Gossen, M. et al. (1995) Science 268: 1766-1769 , PCT Publication No. WO 94/29442, and PCT Publication No. WO 96/01313). Thus, in another aspect, the present invention provides for B7-4 or PD- in eukaryotic cells by operably linking B7-4 or PD-1 DNA to an inducible eukaryotic promoter. A recombinant expression vector that enables inducible expression of one protein is provided.

  The present invention further provides a recombinant expression vector comprising a DNA molecule of the present invention cloned into an expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a manner that allows expression of an RNA molecule that is antisense to B7-4 or PD-1 mRNA (by transcription of the DNA molecule). . A regulatory sequence operably linked to a nucleic acid cloned in an antisense orientation that directs the continuous expression of antisense RNA molecules in various cell types, eg selected by viral promoters and / or enhancers Regulatory sequences can be selected that direct constitutive, tissue-specific or cell-type specific expression of the antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which the antisense nucleic acid is produced under the control of a highly efficient regulatory region, and its activity can be determined by the cell type into which the vector has been introduced. . See Weintraub, H. et al. (1986) “Antisense RNA as a molecular tool for genetic analysis” Review-Trends in Genetics, Volume 1 (1) for a discussion of gene expression regulation using antisense genes. .

  Furthermore, the present invention relates to a host cell into which the recombinant expression vector of the present invention has been introduced. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It should be understood that the above terms encompass not only the particular subject cell, but also the progeny or potential progeny of such cell. The progeny may not be virtually identical to the parent cell because certain modifications may be made in subsequent generations due to mutations or environmental effects, but still fall within the scope of the term used herein. The

  The host cell can be prokaryotic or eukaryotic. For example, B7-4 or PD-1 protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells such as Chinese hamster ovary cells (CHO) or COS cells. Other suitable host cells are known to those skilled in the art.

  Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” include exogenous, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran mediated transfection, lipofection (gene transfer via lipid vesicles) or electroporation. Various art-recognized techniques for introducing nucleic acids (eg, DNA) into host cells are intended to be included. Suitable transformation or transfection methods for host cells are described by Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989), And can be found in other laboratory manuals.

  In order to perform stable transfection of mammalian cells, it is known that depending on the expression vector and transfection technique used, only a small fraction of cells can integrate foreign DNA into their genome. In order to identify and select these components, a gene that encodes a selectable marker (eg, resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those that confer resistance to drugs such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding B7-4 or PD-1 protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (eg, cells into which the selectable marker gene has been incorporated survive and other cells die).

  The host cells of the invention, such as cultured prokaryotic or eukaryotic host cells, can be used for the production (ie, expression) of B7-4 or PD-1 protein. Therefore, the present invention further provides a method for producing B7-4 or PD-1 protein using the host cell of the present invention. One aspect of the above method is that the host cell of the present invention (introduced with a recombinant expression vector encoding B7-4 or PD-1 protein) is suitably used so that B7-4 or PD-1 protein is produced. Culturing in a suitable culture medium. In another embodiment, the method further comprises isolation of B7-4 or PD-1 protein from the medium or the host cell.

  Certain host cells can also be used for the production of non-human transgenic animals. For example, in one embodiment, the host cell is a fertilized oocyte or embryonic stem cell into which a B7-4 or PD-1 coding sequence has been introduced. Then, by using the host cell, a non-human transgenic animal in which an exogenous B7-4 or PD-1 sequence is introduced into the genome or a homologous recombination in which an endogenous B7-4 or PD-1 sequence is modified Animals can be manufactured. The animals are useful for studying the function and / or activity of B7-4 or PD-1 polypeptide and for identifying and / or evaluating modulators of B7-4 or PD-1 activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent, such as a rat or mouse, in which one or more of the animal's cells are transgenes. (Transgene) is included. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens and amphibians. A transgene is an exogenous DNA that is integrated into the genome of a cell from which the transgenic animal develops and remains in the genome of a mature animal, thereby causing one or more cell types or tissues of the transgenic animal Expression of the encoded gene product in is directed. A “homologous recombinant animal” as used herein is a non-human animal, preferably a mammal, more preferably a mouse, in which the endogenous B7-4 or PD-1 gene is Have been modified by homologous recombination between endogenous genes and exogenous DNA molecules introduced into a single cell of the animal, such as an embryonic cell of the animal.

  In transgenic foster animals, for example, by introducing a nucleic acid molecule encoding B7-4 or PD-1 into the male pronucleus of a fertilized oocyte by microinjection or retroviral infection, It can be produced by developing an oocyte. The B7-4 or PD-1 cDNA sequence of SEQ ID NO: 1, 3, 10 or 11 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a non-human homologue of the human B7-4 or PD-1 gene, such as a mouse or rat B7-4 or PD-1 gene, can be used as a transgene. Alternatively, a B7-4 or PD-1 gene homologue, for example, another B7-4 or PD-1 family member is replaced by a B7-4 or PD-1 family cDNA of SEQ ID NO: 1, 3, 10 or 11 Isolated based on hybridization with the sequence (further detailed in subsection I above) and can be used as a transgene. In addition, the transgene expression efficiency can be increased by including an intron sequence and a polyadenylation signal in the transgene. A tissue-specific regulatory sequence (s) is operably linked to a B7-4 or PD-1 transgene to direct expression of B7-4 or PD-1 protein to specific cells. Can do. Methods for producing transgenic animals, particularly animals such as mice, for example, by embryo manipulation and microinjection are already routine in the art, for example, US Pat. Nos. 4,736,866 and 4,487,0009, both by Leder et al. U.S. Pat. No. 4,873,191 to Wagner et al. And Hogan, B. Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1986). Similar methods are used for the production of other transgenic animals. A transgenic founder animal can be identified based on the presence of a B7-4 or PD-1 transgene in its genome and / or expression of B7-4 or PD-1 mRNA in an animal tissue or cell. The transgenic founder animal can then be used to produce additional animals with the transgene. In addition, transgenic animals with transgenes encoding B7-4 or PD-1 protein can be further bred to other transgenic animals with other transgenes.

  In order to produce a homologous recombinant animal, it contains at least a part of the B7-4 or PD-1 gene, and a deletion, addition or substitution is introduced therein, so that the B7-4 or PD-1 gene Modifications are made, eg, functionally disrupted vectors. The B7-4 or PD-1 gene can be a human gene (eg, SEQ ID NO: 1, 3, 10 or 11), but more preferably is a non-human homologue of the human B7-4 or PD-1 gene ( Eg, cDNA isolated by stringent hybridization with the nucleotide sequence of SEQ ID NO: 1, 3, 10 or 11. For example, by using the mouse B7-4 or PD-1 gene, a homologous recombination vector suitable for modifying the endogenous B7-4 or PD-1 gene in the mouse genome can be constructed. In a preferred embodiment, the vector is designed such that upon homologous recombination, the endogenous B7-4 or PD-1 gene is disrupted (ie, also referred to as a “knockout” vector, which no longer encodes a functional protein). ) Alternatively, upon homologous recombination, the endogenous B7-4 or PD-1 gene is mutagenized or otherwise modified, but the vector can still be designed to encode a functional protein (eg, By altering the upstream regulatory region, the expression of endogenous B7-4 or PD-1 protein can be altered). In a homologous recombination vector, the additional nucleic acid sequence of the B7-4 or PD-1 gene is flanked at both 5 ′ and 3 ′ ends of the modified portion of the B7-4 or PD-1 gene. Homologous recombination can be performed between the responsible endogenous B7-4 or PD-1 gene and the endogenous B7-4 or PD-1 gene in embryonic stem cells. The additional flanking B7-4 or PD-1 nucleic acid sequence is of sufficient length for effective homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both 5 ′ and 3 ′ ends) are included in the vector (eg, Thomas, KR and Capecchi, MR (for descriptions of homologous recombination vectors) 1987) Cell 51: 503). The vector is introduced into an embryonic stem cell line (eg, by electroporation), and the introduced B7-4 or PD-1 gene is homologously recombined with the endogenous B7-4 or PD-1 gene. (See, eg, Li, E. et al. (1982) Cell 69: 915). The selected cells are then injected into undifferentiated germ cells of animals (eg, mice) to form aggregate chimeras (eg, Bradley, A., Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, edited by Robertson, EJ). (See IRL, Oxford, 1987) pages 113-152). The chimeric embryo can then be transferred into a suitable pseudopregnant female foster animal and the embryo delivered. By using progeny with homologous recombinant DNA in germ cells, all cells of the animal can produce animals containing the homologous recombinant DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described in Bradley, A. (1991) Curr. Opin. Biotechnol. 2: 823-829 and Le Mouellec et al., PCT International Publication No. WO 90/11354, Smithies et al. No. WO 91/01140, Zijlstra et al. WO 92/0968, and Berns et al. WO 93/04169.

  In addition to the foregoing, one skilled in the art will recognize that other methods known in the art for homologous recombination can also be applied to the present invention. Enzyme-assisted site-specific integration systems are known in the art and can be applied to integrate a DNA molecule at a predetermined location in a second target DNA molecule. Examples of such enzyme-assisted integration systems include Cre recombinase-lox target systems (eg, Baubonis, W. and Sauer, B. (1993) Nucl. Acids Res. 21: 2025-2029, and Fukushige, S. and Sauer, B. (1992) Proc. Natl. Acad. Sci. USA 89: 7905-7909) and FLP recombinase-FRT target systems (eg, Dang, DT and Perrimon, N. (1992) Dev. Genet. 13). : 367-375, and Fiering, S. et al. (1993) Proc. Natl. Acad. Sci. USA 90: 8469-8473). Tetracycline-regulated inducible homologous recombination systems such as those described in PCT Publication No. WO94 / 29442 and PCT Publication No. WO96 / 01313 may also be used.

  For example, in another embodiment, a transgenic non-human animal can be produced that includes a system selected to allow transgene expression regulation. An example of such a system is the cre / loxP recombinase system of bacteriophage P1. For a description of the cre / loxP recombinase system, see, eg, Lakso et al. (1992) Proc. Natl. Acad. Sci. USA 89: 6232-6236. Another example of a recombinase system is the Saccharomyces cerebvisiae FLP recombinase system (O'Gorman et al. (1991) Science 251: 1351-1355). When the cre / loxP recombinase system is used to regulate transgene expression, animals containing transgenes encoding both the Cre recombinase and the selected protein are required. The animal is, for example, a “double” transgenic animal by crossing two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase. Can be provided through construction.

In addition, clones of transgenic animals other than humans described herein are the methods described in Wilmut, I. et al. (1997) Nature 385: 810-813 and PCT International Publication Nos. WO97 / 07668 and WO97 / 07669. Can be manufactured. Briefly, cells from a transgenic animal, such as somatic cells, can be isolated and induced to exit the growth cycle and enter the GO phase. The quiescent cells can then be fused to enucleated oocytes from the same animal from which the quiescent cells were isolated, for example through the use of electrical pulses. The reconstructed oocyte is then cultured so that it develops into morula or blast and then transferred to a pseudopregnant female foster animal. The offspring born from this female foster animal becomes a clone of the animal from which cells, eg, somatic cells, were isolated.

V. Pharmaceutical compositions B7-4 or PD-1 modulators (eg, B7-4 or PD-1 inhibitors or stimulators such as B7-4 or PD-1 nucleic acid molecules, proteins, antibodies or B7-4 or The compounds identified as modulators of PD-1 activity and / or expression or modulators of the interaction between B7-4 and PD-1) can be incorporated into pharmaceutical compositions suitable for administration. The composition typically comprises a nucleic acid molecule, protein or antibody and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” includes all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with pharmaceutical administration. And The use of such media and agents for pharmaceutically active substances is known in the art. Except where the conventional medium or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds can also be incorporated into the compositions.

  A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, eg intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (topical), transmucosal and rectal administration. Solutions or suspensions used for parenteral, intradermal or subcutaneous application are composed of the following components: sterile diluents such as water for injection, saline, non-volatile oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents Antibacterial agents such as benzyl alcohol or methyl paraben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetate, citrate or phosphate and tonicity modifiers such as sodium chloride or dextrose Can be included. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

  Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL ™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Long-term absorption of the injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

  Sterile injectable solutions contain the required amount of active compound (eg, B7-4 or PD-1 protein or anti-B7-4 or PD-1 antibody) in a suitable solvent along with one or a combination of the ingredients listed above And then, if necessary, sterile filtered. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the production of sterile injectable solutions, the preferred production methods are vacuum drying and lyophilization, which produces a powder consisting of the desired additional ingredients in addition to the active ingredients from the pre-sterilized filtered solution .

  Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be made with a fluid carrier used as a mouthwash, where the compound in the fluid carrier is applied orally, swished, exhaled or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches and the like may be any of the following components or compounds of similar nature: binders such as microcrystalline cellulose, tragacanth gum or gelatin, excipients such as starch or lactose, disintegrants such as alginic acid Primogel or corn starch, lubricants such as magnesium stearate or Sterotes, lubricants such as colloidal silicon dioxide, sweeteners such as sucrose or saccharin, or flavorings such as peppermint, methyl salicylate or orange flavors obtain.

  For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

  Systemic administration can also be performed by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

  The compounds can also be prepared in suppositories for rectal delivery (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas.

  In one embodiment, the modulator agent is manufactured using a carrier that protects the compound from rapid elimination from the body, such as a controlled release formulation comprising an implant and a microencapsulated delivery system. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. The method for producing the above formulation will be apparent to those skilled in the art. These materials can also be purchased from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to cells infected with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US Pat. No. 4,522,811.

  It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. The unit dosage form used herein includes physically independent units suitable as unit dosages for the subject to be treated, each unit with a pharmaceutical carrier required to produce the desired therapeutic effect. A predetermined amount of active compound calculated in Details regarding the unit dosage forms of the present invention are inevitably directly determined by the specific characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the limitations inherent in the art of formulating such active compounds for the treatment of individuals. The

  Toxicity and therapeutic efficacy of the above compounds are measured by standard pharmaceutical methods in cell cultures or experimental animals to determine, for example, LD50 (lethal dose for 50% of the population) and ED50 (therapeutically effective dose in 50% of the population). obtain. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit large therapeutic indices are preferred. When compounds that exhibit toxic side effects can be used, carefully design delivery systems that target the compounds to affected tissue sites to reduce side effects by minimizing potential damage to uninfected cells. Should be done.

  Data obtained from cell culture assays and animal studies can be used to formulate a range of doses for use in humans. The dose of the compound lies preferably within a circulating concentration range that includes the ED50 with little or no toxicity. The dose may vary within this range depending on the dosage form used and the route of administration used. For compounds used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. Formulating the dose in an animal model provides a circulating plasma concentration range that includes the IC50 (ie, the concentration of the test compound that achieves half-maximal inhibition of symptoms) measured in cell culture. By using the above information, useful doses in humans can be more accurately determined. Plasma levels can be measured, for example, by high performance liquid chromatography.

  The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, topical administration (see US Pat. No. 5,328,470) or stereotaxic injection (see, eg, Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3054-3057). The pharmaceutical formulation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is embedded. Alternatively, where a complete gene delivery vector can be produced intact from recombinant cells (eg, a retroviral vector), the pharmaceutical formulation can include one or more cells that produce the gene delivery system.

  The pharmaceutical composition may be enclosed in a pack or dispenser together with a container, instructions for administration.

VI. Uses and Methods of the Invention B7-4 and / or PD-1 modulator agents described herein, such as nucleic acid molecules, proteins, protein homologues and antibodies, may be used in one or more of the following methods. Obtain: a) methods of treatment, for example by up- or down-modulation of the immune response, b) screening assays, c) predictive medicine (eg, diagnostic assays, prognostic tests, monitor clinical trials, and pharmacogenetics). An isolated nucleic acid molecule of the invention can be expressed, for example, by expressing a B7-4 or PD-1 protein (eg, by a recombinant expression vector in a host cell for gene therapy applications), as detailed below. Used to detect genetic alterations in B7-4 or PD-1 mRNA (eg, in a biological sample) or B7-4 or PD-1 gene and modulate B7-4 or PD-1 activity obtain. B7-4 or PD-1 protein can be used in the treatment of diseases characterized by insufficient or excessive production of B7- or PD-1 inhibitors. Further, the B7-4 or PD-1 protein may be screened for naturally occurring B7-4 or PD-1 binding partners, screened for agents or compounds that modulate B7-4 or PD-1 activity, and B7-4 or PD-1 Used for the treatment of diseases characterized by insufficient or excessive production of -1 protein or production of B7-4 or PD-1 protein forms having reduced or abnormal activity compared to B7-4 or PD-1 wild type protein Can be done. Further, the anti-B7-4 or PD-1 antibodies of the present invention can detect and isolate B7-4 or PD-1 protein, modulate the bioavailability of B7-4 or PD-1 protein, and for example B7 Can be used to modulate B7-4 or PD-1 activity by modulating the interaction of -4 and PD-1.

A. Treatment method:
The present invention provides methods for the prophylactic and therapeutic treatment of subjects at risk (or susceptible) or suffering from diseases associated with abnormal B7-4 or PD-1 expression or activity.

1. Prophylaxis In one aspect, the invention administers to a subject an agent that modulates B7-4 or PD-1 polypeptide or B7-4 or PD-1 polypeptide expression or at least one B7-4 or PD-1 activity. To prevent a disease or condition associated with abnormal B7-4 or PD-1 expression or activity in a subject. Subjects at risk of disease induced by or resulting from aberrant B7-4 or PD-1 expression or activity can be identified, for example, by any or a combination of the diagnostic or prognostic tests described herein . Administration of a prophylactic agent can occur before the onset of symptoms characterized by an abnormal form of B7-4 or PD-1 to prevent the disease or disorder or otherwise slow its progression. Depending on the type of B7-4 or PD-1 abnormality or condition, for example, a B7-4 or PD-1 polypeptide, a B7-4 or PD-1 agonist or a B7-4 or PD-1 antagonist agent treats the subject Can be used for. Appropriate agents can be determined based on clinical indications and can be confirmed, for example, using the screening assays described herein.

2. Therapeutic Methods Another aspect of the invention relates to a method of modulating B7-4 or PD-1 expression or activity for therapeutic purposes. B7-4 has been shown to inhibit costimulation and proliferation of activated immune cells and transmit an inhibitory signal to immune cells via PD-1. Thus, by modulating the activity and / or expression of B7-4 or PD-1 and the interaction between B7-4 and PD-1, the immune response can be modulated. In embodiments where B7-4 binds to a costimulatory receptor, upregulation of B7-4 activity induces upregulation of the immune response, and downregulation of B7-4 activity induces downregulation of the immune response. You should understand that. In embodiments where B7-4 binds to an inhibitory receptor, upregulation of B7-4 activity induces downregulation of the immune response, and downregulation of B7-4 activity induces upregulation of the immune response. In a preferred embodiment, B7-4 binds to an inhibitory receptor. In a particularly preferred embodiment, B7-4 binds to PD-1.

  In the modulation method of the present invention, the cells are treated with a modulator of B7-4 or PD-1 polypeptide, eg, an agent that modulates the expression or activity of B7-4 and / or PD-1, or the interaction between B7-4 and PD-1. Contact with an agent that modulates the action.

  Agents that modulate B7-4 or PD-1 protein activity are those described herein, such as nucleic acids or protein molecules of B7-4 or PD-1 protein, naturally occurring target molecules (eg, B7- 4 for PD-1 or PD-1, B7-4), B7-4 or PD-1 antibody, B7-4 or PD-1 agonist or antagonist, B7-4 or PD-1 agonist or There are peptidomimetics of antagonists, or other small molecules.

  Agents that modulate B7-4 or PD-1 expression are, for example, antisense nucleic acid molecules, triplex oligonucleotides, or ribozymes or recombinant vectors for B7-4 or PD-1 protein expression. For example, oligonucleotides complementary to the region surrounding the B7-4 or PD-1 polypeptide translation initiation site can be synthesized. The synthesis of B7-4 or PD-1 polypeptide can be blocked by adding one or more antisense oligonucleotides to the cell culture medium, typically at 200 μg / ml, or administering to a patient. Antisense oligonucleotides are absorbed by cells and block translation by hybridizing to B7-4 or PD-1 mRNA. Alternatively, oligonucleotides that block DNA unwinding and transcription by binding to double stranded DNA to form a triplex construct can be used. Either result will block the synthesis of B7-4 or PD-1 polypeptide. When PD-1 expression is modulated, preferably such modulation is done by means other than knocking out the PD-1 gene.

  Agents that modulate expression also modulate the total amount of PD-1 or B7-4 activity in the cell due to the fact that they control the amount of PD-1 or B7-4 in the cell.

  In one embodiment, the agent that stimulates B7-4 inhibitory activity or PD-1 inhibitory activity is an agonist of B7-4 or PD-1. Examples of such agents include active B7-4 or PD-1 proteins and nucleic acid molecules encoding B7-4 or PD-1 polypeptides introduced into cells.

  In another embodiment, the agent inhibits B7-4 costimulatory or inhibitory activity or PD-1 inhibitory activity and is an antagonist of B7-4 or PD-1. Examples of such agents include antisense B7-4 or PD-1 nucleic acid molecules, anti-B7-4 or PD-1 antibodies, soluble inactivated forms of B7-4 or PD-1 molecules, and B7-4 or PD- There is one inhibitor.

  These modulator agents can be administered in vitro (eg, by contacting a cell with the agent) or alternatively in vivo (eg, by administering the agent to a subject). As such, the present invention relates to diseases or disorders that benefit from modulation of B7-4 or PD-1 protein, such as up or down regulation of immune response, or B7-4 or PD-1 protein or nucleic acid. Methods of treating individuals suffering from diseases characterized by aberrant expression or activity of the molecule are provided. In one aspect, the method comprises an agent (eg, an agent identified by a screening assay described herein) or an agent that modulates B7-4 or PD-1 expression or activity (eg, upregulation or downregulation). Administer combination. In another embodiment, the method administers a B7-4 or PD-1 protein or nucleic acid molecule as a therapy that compensates for reduced or abnormal B7-4 or PD-1 expression or activity.

  Stimulation of B7-4 or PD-1 activity is a situation where B7-4 or PD-1 is abnormally down-regulated and / or where high B7-4 or PD-1 activity appears to have an advantageous effect Then it is desirable. Similarly, inhibition of B7-4 or PD-1 activity may indicate that B7-4 or PD-1 is abnormally up-regulated and / or that low B7-4 or PD-1 activity has an advantageous effect. Desirable in situations where it seems. One skilled in the art will recognize that in embodiments where B7-4 binds to a costimulatory one, stimulation of B7-4 and PD-1 have the opposite effect on immune cell costimulation and thus immune response. It should be. In such a case, when stimulation of the activity of one molecule is desired, suppression of the activity of other molecules is desirable.

  Examples of agents (B7-4 antagonists) used for down-modulation of B7-4 include (for example) antisense molecules, antibodies that recognize B7-4, one of B7-4 and its natural receptor in immune cells. There are compounds that block one interaction (eg, soluble monovalent B7-4 molecule, and soluble form of B7-4 ligand or compounds identified in a subject screening assay). Examples of agents (PD-1 antagonists) used for PD-1 down-modulation include (for example) antibodies that bind to an antisense molecule, PD-1, but do not transmit an inhibitory signal to immune cells (“inactive Non-activating antibody), and soluble forms of PD-1.

  Examples of agents (B7-4 agonists) used for B7-4 up-modulation include (for example) nucleic acid molecules encoding B7-4 polypeptides, multivalent forms of B7-4, expression of B7-4 Compounds that enhance, and cells that express B7-4. Examples of drugs (PD-1 agonists) used for PD-1 up-modulation include (for example) antibodies that transmit inhibitory signals via PD-1, compounds that promote PD-1 expression, PD There are nucleic acid molecules encoding -1, and a form of B7-4 that transmits signals via PD-1.

3. Down-regulation of immune response by modulation of B7-4 or PD-1 According to the present invention, the immune response is down-regulated by up-regulating the inhibitory function of B7-4 polypeptide or down-regulating its costimulatory function There are many aspects. Down-regulation can be in a form that inhibits or blocks an immune response that has already occurred, or can include blocking the induction of an immune response.

  The function of activated immune cells can be inhibited by down-regulation of immune cell responses, by induction of specific anergy in immune cells, or both.

  For example, block B7-4 interaction with anti-B7-4 antibody or B7-4 polypeptide (eg, soluble monomeric form of B7-4 polypeptide, eg B7-4-Ig) and / or costimulatory receptors. By using anti-B7-4 antibodies, costimulatory signals can be inhibited, thereby down-modulating the immune response.

  Further, in embodiments where B7-4 binds to an inhibitory receptor, the immune response is reduced by using a form of B7-4 that binds to the inhibitory receptor, eg, multivalent B7-4 on the cell surface. Can be modulated.

  Similarly, the PD-1 pathway can also be stimulated by using drugs to down regulate the immune response. Inhibition of interaction of B7-4 with stimulatory receptors in immune cells (eg, by using soluble forms of PD-1 and / or CTLA4) or activation of PD-1 (eg, cross-linking with PD-) Can be used to provide a negative signal to immune cells.

  In one aspect of the invention, the activating antibody used to stimulate PD-1 activity is a bispecific antibody. For example, such antibodies can include a PD-1 binding site and another binding site that targets cell surface receptors in immune cells such as T cells, B cells or bone marrow cells. In one embodiment, such an antibody, in addition to containing a PD-1 binding site, further comprises a molecule adjacent to an activating or inhibitory receptor, such as a B cell antigen receptor, a T cell antigen receptor or an Fc receptor. By including binding sites that bind, molecules can be targeted to specific cell populations. For example, CD3 antigen, T cell receptor chain, LFA-1, CD2, CTLA-4, immunoglobulin, B cell receptor, Ig alpha, Ig beta, CD22 or Fc receptor can be used. Such antibodies (or other bispecific agents) are recognized in the art and can be produced, for example, as described herein. This selection of the second antigen for the bispecific antibody gives flexibility in the selection of the cell population targeted for inhibition.

  In another aspect, co-ligation of activating or inhibitory receptors in PD-1 and cells can facilitate the generation of negative signals via PD-1. The co-ligation reaction can be accomplished, for example, by the use of bispecific antibodies described herein that have specificity for both a bispecific agent, such as PD-1 and a molecule associated with the receptor. In another embodiment, the use of a multivalent form of an agent that transmits a negative signal through PD-1 can facilitate the transmission of the negative signal through PD-1, for example displayed on a bead or surface. There are drugs. In another aspect, such multivalent agents include two specificities to allow co-ligation of PD-1 and a receptor or receptor-associated molecule (eg, a bead containing anti-CD3 and B7-4). Can be achieved.

  Agents that block or inhibit the interaction of B7-4 with costimulatory receptors (eg, soluble forms of B7-4 or blocking antibodies to B7-4) and agents that promote B7-4 transmission inhibitory signals or PD- PD-1 agonists that activate 1 (eg, PD-1 activating antibodies or PD-1 activating small molecules) are added to in vitro assays where they inhibit immune cell proliferation and / or effector function Can be confirmed by the ability to induce anergy. For example, cells can be cultured in the presence of an agent that stimulates signal transduction via an activated receptor. By using some cell activation readout methods recognized in the art, cell proliferation or effector function (eg, antibody production, cytokine production, phagocytosis) can be measured in the presence of an active agent. The ability of a test agent to block this activation can be readily determined by measuring the agent's ability to reduce the proliferation or effector function being measured.

  In one aspect of the invention, tolerance is induced against a specific antigen by co-administering the antigen with a PD-1 agonist. For example, tolerance can be induced for specific proteins. In one aspect, an immune response against an allergen or foreign protein in which an immune response is undesirable may be inhibited. For example, patients receiving factor VIII often produce antibodies against this clot forming factor. An agent that blocks a B7-4 signaling costimulatory signal or an agent that stimulates a PD-1 signaling inhibitory signal is combined with recombinant factor VIII (or physically bound to factor VIII, eg, by cross-linking). By administration, down modulation can be induced.

  In one embodiment, in a immune cell by using a fusion protein wherein the B7-4 first peptide is fused to a second peptide having the activity of another B lymphocyte antigen (eg, B7-1 or B7-2). The interaction of costimulatory receptors with B7-4 can be blocked and the immune response can be down-modulated. Alternatively, two separate peptides (eg, B7-2 and / or B7-1 and B7-4 polypeptides), or a combination of blocking antibodies (eg, antibodies to B7-4 polypeptides and anti-B7-2) And / or B7-1 monoclonal antibodies) can be combined as a single composition or administered separately (simultaneously or sequentially) to down-regulate the immune cell transfer immune response in the subject. In addition, the use of therapeutically effective amounts of one or more peptides having B7-4 polypeptide activity along with B7-1 and / or B7-1 activity in combination with other down-regulation reagents can affect the immune response. obtain. Examples of other immune modulation reagents include antibodies that block costimulatory signals (eg, against CD28, ICOS), antibodies that activate inhibitory signals via CTLA4, and / or other immune cell markers ( Examples are anti-CD40, anti-CD40 ligand or anti-cytokine) antibodies, fusion proteins (eg, CTLA4-Fc, PD-1-Fc) and immunosuppressive agents (eg, rapamycin, cyclosporin A or FK506).

  B7-4 and / or PD-1 peptides may also be useful in the construction of therapeutic agents that block immune cell function by cell destruction. For example, conjugating a portion of a B7-4 or PD-1 polypeptide to a toxin can produce a cytotoxic agent that can induce destruction of the cell to which it is bound.

  To produce a cytotoxic agent, the polypeptides of the invention can be conjugated or functionally linked to a toxin using techniques known in the art, such as cross-linking or recombinant DNA techniques. The production of immunotoxins is generally well known in the art (see, eg, US Pat. Nos. 4,340,535 and EP 44167, both of which are hereby incorporated by reference). Numerous types of disulfide bonds are known that can be used effectively to conjugate a toxin moiety to a polypeptide, including linkers. In one embodiment, linkers containing sterically hindered disulfide bonds are preferred because of greater in vivo stability, which prevents release of the toxin moiety prior to attachment at the site of action.

  A wide variety of toxins are known that can be conjugated to the polypeptides or antibodies of the present invention. Examples include many useful plant, fungal or bacterial derived toxins such as various A chain toxins, especially ricin A chain, ribosome inactivating proteins such as saporin or gelonin, alpha, -sarcin, aspergillin, restrictocin, There are ribonucleases such as placental ribonuclease, angiogenic, diphtheria toxin, and Pseudomonas exotoxin. A preferred toxin moiety for use in connection with the present invention is toxin A chain, deglycosylated A chain that has been treated to modify or remove carbohydrate residues (US Pat. No. 5,776,427).

  By injecting one or a combination of the above cytotoxic agents (eg, B7-4 lysine alone or B7-2 lysine or a combination with B7-1 lysine) into a patient, in particular, B7 with more abundant activated immune cells. In light of the fact that -4 ligand is expressed, immune cell death can also be induced. For example, because PD-1 is induced on the surface of activated lymphocytes, antibodies against PD-1 are either cytotoxic by Fc-R-dependent mechanisms or cytotoxic agents (eg, lysine, saporin or calicheamicin). Can be used to target the removal of these specific cells by conjugation. In one embodiment, the antibody toxin can be a bispecific antibody. Such bispecific antibodies are useful for targeting specific cell populations using, for example, markers found only in certain types of cells, such as TCR, BCR or FcR molecules.

  Downregulation or blocking of B7-4 polypeptide costimulatory function or activation of B7-4 or PD-1 inhibitory function (eg, by stimulating the negative signaling function of PD-1) is e.g. tissue It is useful for downmodulating the immune response in skin and organ transplant situations, graft-versus-host disease (GVHD), or autoimmune diseases such as systemic lupus erythematosus and multiple sclerosis. For example, blockage of immune cell function reduces tissue destruction in tissue transplantation. Typically, in the case of tissue transplantation, transplantation rejection is initiated by an immune reaction that destroys the transplant after immune cells are recognized as foreign. Molecules that block or block the interaction of costimulatory receptor (s) and B7 molecules in immune cells (eg, soluble monomeric forms of B7-4 or PD-1 polypeptide), either alone or at the time of transplantation When administered with other down-modulating agents, the generation of costimulatory signals can be prevented. Furthermore, inhibition of B7-4 costimulatory signals, or promotion of B7-4 or PD-1 inhibitory signals may also be sufficient to anergy the immune cells, thereby inducing tolerance in the subject. . Induction of long-term tolerance by blocking B7-4 transducing costimulatory signals can avoid the need for repeated administration of these blocking reagents.

  It may be desirable to block the co-stimulatory function of other molecules in order to achieve sufficient immunosuppression or tolerance in the subject. For example, it may be desirable to block the function of B7-1 and B7-4, B7-2 and B7-4, or B7-1 and B7-2 and B7-4, and these antigens before or at the time of transplantation Soluble forms of peptides having the respective activity or combinations of blocking antibodies against these antigens can be administered (separately or together in a single composition). Alternatively, it may be desirable to promote the inhibitory activity of B7-4 or PD-1 and block the costimulatory activity of B7-1 and / or B7-2. Other down-modulating agents that can be used in connection with the down-modulation methods of the invention include, for example, agents that transmit inhibitory signals via CTLA4, soluble forms of CTLA4, antibodies that activate inhibitory signals via CTLA4, and other immunity There are blocking antibodies to soluble forms of cell markers or other receptor ligand pairs (eg, agents that disrupt the interaction between CD40 and CD40 ligand (eg, anti-CD40 ligand antibodies)), antibodies to cytokines or immunosuppressive agents. In another aspect, optimal blocking activity can be achieved by administering a combination of at least two different B7-4 antibodies.

  For example, blocking B7-4 polypeptide costimulation or activating B7-4 or PD-1 inhibitory function is also useful in the treatment of autoimmune diseases. Many autoimmune diseases are the result of inappropriate activation of immune cells that are reactive to self tissue and promote the production of cytokines and autoantibodies involved in the disease pathology. By preventing the activation of autoreactive immune cells, the symptoms of the disease can be reduced or eliminated. Costimulatory Receptor and B7 Molecule Receptor: Administering reagents that block immune cell costimulation by disrupting ligand interactions prevents immune cell activation and is involved in the disease process It is useful to block the production of the resulting autoantibodies or cytokines. Thus, agents that promote the inhibitory function of B7-4 or PD-1 can induce antigen-specific tolerance of autoreactive immune cells, resulting in long-term relief of the disease. The efficacy of a reagent in preventing or reducing autoimmune disease can be measured using some well-characterized animal model of human autoimmune disease. Examples include murine experimental autoimmune encephalitis, systemic lupus erythematosus in MRL / lpr / lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia (See Paul, Fundamental Immunology, Raven Press, New York, 1989, pages 840-856).

  Prevention of immune cell activation is therapeutically useful in the treatment of allergies and allergic reactions, eg, by inhibiting IgE production. By administering an agent that promotes B7-4 or PD-1 inhibitory function to an allergic subject, an immune cell transmitted allergic response in the subject can be blocked. Activation of PD-1 polypeptide may also be useful in the treatment of allergies. Inhibition of B7-4 costimulation of immune cells or stimulation of the B7-4 or PD-1 inhibition pathway can be achieved by exposure to allergens with appropriate MHC molecules. Allergic reactions can be systemic or local in nature due to the allergen entry pathway and the pattern of IgE precipitation in mast or basophil cells. That is, inhibition of a local or systemic immune cell transmission allergic response can be achieved by inhibiting an agent that blocks the interaction of B7-4 with a costimulatory receptor or an agent that promotes the inhibitory function of B7-4 or PD-1. This can be done by administering a sex form.

  Inhibition of immune cell activation by blocking B7-4 costimulatory activity or stimulation of PD-1 inhibitory activity may also be therapeutically important in viral infections of immune cells. For example, in the case of acquired immune deficiency syndrome (AIDS), viral replication is stimulated by immune cell activation. Blocking the B7-4 / costimulatory receptor interaction or stimulating the B7-4 or PD-1 inhibitory function can induce inhibition of viral replication, thereby improving the course of AIDS. Downregulation of the immune response by stimulating the interaction of B7-4 activity or B7-4 with its natural binding partner (s), eg PD-1, may also be useful to promote pregnancy persistence. B7-4 is normally highly expressed in the placental trophoblast, the cell layer that forms the interface between the maternal and fetus, and may play a role in preventing maternal rejection of the fetus. Women at risk of spontaneous abortion due to immunological rejection of the embryo or fetus (eg, experience of spontaneous abortion if confirmed by screening for B7-4 activity as described in the “Prognostic Test” section) May be treated with an agent that stimulates the activity of B7-4 or its natural binding partner (s), such as interaction with PD-1.

  Downregulation of the immune response by stimulating B7-4 activity or its original binding partner (s), eg, B7-4 interaction with PD-1, also treats autoimmune seizures in autologous tissues Can be useful to do. For example, B7-4 is normally highly expressed in the heart and protects the heart from autoimmune attacks. This is evidenced by the fact that Balb / cPD-1 knockout mice exhibit severe autoimmune attacks in the heart with thrombosis. That is, a condition induced or exacerbated by an autoimmune attack (eg, heart disease, myocardial infarction or atherosclerosis in this example) is associated with B7-4 activity or its original binding partner, eg PD-1. It can be improved or improved by increasing the binding of B7-4. Thus, conditions exacerbated by autoimmune stroke, such as autoimmune disease (and eg heart disease, myocardial infarction and atherosclerosis) by stimulating B7-4 activity or B7-4 interaction with B7-4 It is also included within the scope of the present invention.

4). Upregulation of immune response Upregulation of B7-4 costimulatory activity or inhibition of PD-1 or B7-4 inhibitory activity is also therapeutically useful as a means of upregulating immune response. Upregulation of the immune response can be in a form that enhances an existing immune response or induces a primary immune response. For example, stimulating an immune response by stimulating B7-4 costimulatory activity or inhibiting B7-4 or PD-1 inhibitory activity is useful in the case of microorganisms such as bacteria, viruses or parasitic infections. For example, in one aspect, a form of B7-4 that promotes a costimulatory signal in immune cells (eg, a multivalent form of B7-4 peptide (eg, a soluble multivalent form or a form expressed on the cell surface)) or Agents that block the interaction of B7-4 with inhibitory receptors or agents that block the transmission of inhibitory signals by PD-1, such as non-activated antibodies to PD-1, can upregulate antibodies and cell-mediated responses. However, it is therapeutically useful in situations where it is beneficial because it allows rapid or complete removal of the virus. These include viral skin diseases such as herpes or shingles, in which case the drug can be delivered topically to the skin. Furthermore, systemic viral diseases such as influenza, common colds and encephalitis can be alleviated by systemic administration of the drug.

  In some cases, in order to further increase the immune response, other agents that up-regulate the immune response, such as other forms of B7 family components that signal through a costimulatory receptor may be further administered. It may be desirable.

  Alternatively, an agent that removes immune cells from the patient and blocks the interaction of a form of B7-4 or an inhibitory receptor with B7-4 that promotes costimulatory signals in the immune cells, or PD-1 The immune response can be enhanced in infected patients by contacting in vitro immune cells with agents that block the transmission of inhibitory signals by and reintroducing the in vitro stimulated immune cells into the patient. In another embodiment, a method for enhancing an immune response isolates infected cells, such as virus-infected cells, from a patient, such that the cells express all or part of the B7-4 molecule on their surface. They are transfected with a nucleic acid molecule encoding a form of B7-4 that binds to the body and the transfected cells are reintroduced into the patient. Transfected cells can deliver costimulatory signals to activate immune cells in vivo.

  Various forms of B7-4 that promote costimulatory signals in immune cells, agents that block the interaction of B7-4 with inhibitory receptors, or agents that block the transmission of inhibitory signals by PD-1 Can be used prophylactically in vaccines against other polypeptides, such as polypeptides from pathogens. Immunity against pathogens such as viruses is a form of B7-4 that promotes costimulatory signals in immune cells, or an agent that blocks the interaction of inhibitory receptors with B7-4, or PD in an appropriate adjuvant. It can be induced by inoculating viral proteins with agents that block the transmission of inhibitory signals by -1. Alternatively, vectors containing genes encoding for both a pathogenic antigen and a form of B7-4 that binds to costimulatory receptors can be used for vaccination. Nucleic acid vaccines can be obtained by various means, for example, injection (eg, biolistic injection of DNA-coated gold particles into the epidermis by gene guns that inject particles into the skin using intramuscular, intradermal, or particle accelerators or compressed gas ( Haynes et al., 1996, J. Biotechnol. 44:37)). Alternatively, the nucleic acid vaccine can be administered by non-invasive means. For example, pure or lipid formulated DNA can be delivered to the respiratory system or other targeted locations, such as Peyer's patches by oral delivery of DNA (Schubbert. 1997, Proc. Natl. Acad. Sci. USA 94). : 961). Attenuated microorganisms can be used for delivery to mucosal surfaces (Sizemore et al., 1995, Science, 270: 29).

In one embodiment, one form of a B7-4 polypeptide that transmits a costimulatory signal is, for example, a cell transfected to co-express a B7-4 polypeptide and an MHC class I α chain protein and β 2 microglobulin. Can be administered together with class I MHC proteins to induce T cell activation and provide immunity from infection. For example, pathogens for which vaccines are useful include hepatitis B, hepatitis C, Epstein-Barr virus, cytomegalovirus, HIV-1, HIV-2, tuberculosis, malaria and schistosomiasis.

  In another application, upregulation or promotion of B7-4 costimulatory function is useful for inducing tumor immunity. Tumor specific tolerance in a subject by administering to the subject a tumor cell (eg, sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid molecule encoding the B7-4 antigen Can be overcome. If desired, tumor cells can be transfected to express a combination of B7 polypeptides (eg, B7-1, B7-2, B7-4). For example, tumor cells obtained from a patient can be transfected ex vivo with an expression vector that directs the expression of a B7-4 polypeptide alone or in combination with a peptide having B7-1 activity and / or B7-2 activity. Returning the transfected tumor cells to the patient induces peptide expression on the surface of the transfected cells. Alternatively, gene therapy techniques can be used to target tumor cells for in vivo transfection.

In addition, tumor cells lacking MHC class I or MHC class II molecules or failing to express sufficient amounts of MHC class I or MHC class II molecules may be treated with MHC class I α chain protein and β 2 microglobulin protein or MHC class. By transfecting with a nucleic acid encoding all or part of the IIα chain protein and MHC class II β chain protein (eg, cytoplasm-domain truncated region), the MHC class I or MHC class II protein is expressed on the cell surface. obtain. Expression of an appropriate class I or class II MHC with a peptide having the activity of a B lymphocyte antigen (eg, B7-1, B7-2, B7-4) results in a T cell-mediated immune response against transfected tumor cells Is induced. Optionally, a gene encoding an antisense construct that blocks expression of an MHC class II-related protein, eg, an invariant chain, can also be cotransfected with DNA encoding a B7-4 polypeptide, thereby It can facilitate presentation and induce tumor specific immunity. Expression of B7-1 by B7-negative murine tumor cells has been shown to conduct tumor challenge in mice by inducing T cell-mediated specific immunity with tumor rejection and long-term protection (Chen, L. et al. (1992) Cell 71, 1093-1102, Townsend, SE and Allison, JP (1993) Science 259, 368-370, Baskar, S. et al. (1993) Proc. Natl. Acad. Sci. 90, 5687-5690). That is, induction of an immune cell-mediated immune response in a human subject may be sufficient to overcome tumor specific tolerance in the subject.

  In another embodiment, the immune response is by signaling through a costimulatory receptor that binds to B7-4 or an inhibitory receptor that binds to B7-4 such that pre-existing tolerance is overcome. For example, it can be stimulated by blocking signal generation via PD-1. For example, an immune response against an antigen for which the subject cannot develop a significant immune response, such as an autologous antigen, such as a tumor-specific antigen, blocks the inhibitory activity of PD-1 or the ability of B7-4 to bind to an inhibitory ligand. Can be induced by administering an agent to be treated. For example, in one aspect, the use of soluble PD-1 or soluble B7-4 (PD-1 Fc or B7-4Fc) can promote an immune response against, for example, tumor cells. In one aspect, an autologous antigen, such as a tumor-specific antigen, can be co-administered with an agent that blocks PD-1 inhibitory activity or the ability of B7-4 to bind to an inhibitory ligand. In another embodiment, a neurological disease can be treated by stimulating an immune response against an antigen (eg, an autologous antigen). In another aspect, a PD-1 antagonist can be used as an adjuvant to increase the response to foreign antigens in the active immunization process.

  In yet another aspect, a form of B7-4 that binds to an inhibitory receptor or competes with the binding of B7-4 to a costimulatory receptor (eg, a form of B7-4 that binds to PD-1) Alternatively, the immune response can be up-regulated by inhibiting the production of naturally occurring soluble molecules) using, for example, antisense RNA. For example, in one aspect, production of inhibitory B7-4 molecules by tumor cells can be inhibited to increase anti-tumor immunity.

  In one aspect, immune cells are obtained from a subject ex vivo in the presence of a form of B7-4 that binds a costimulatory molecule or in the presence of an agent that suppresses a B7-4 or PD-1 inhibitory signal. By culturing, the population of immune cells expands. In yet another embodiment, immune cells are then administered to the subject. As is known in the art, immune cells can be stimulated and proliferated in vitro, for example, by providing immune cells with primary activation and costimulatory signals. In addition, proliferation of immune cells can be costimulated by using agents that bind to various forms of B7-4 protein or costimulatory receptors or block signal generation via PD-1. In one aspect, immune cells are cultured ex vivo according to the methods described in PCT application No. WO 94/29436. Co-stimulatory molecules are soluble and can be bound to cell membranes or bound to solid surfaces such as beads.

B. Identification of cytokines modulated by modulation of B7-4 and / or PD-1 By using the B7-4 and PD-1 molecules described herein, modulation of B7-4 and / or PD-1 activity can be achieved. Cytokines can be identified that are produced by immune cells in response or whose production is promoted or inhibited by immune cells. Immune cells expressing PD-1 can be stimulated suboptimally in vitro with a primary activation signal, eg, T cells are stimulated with phorbol esters, anti-CD3 antibodies or preferably antigens associated with MHC class II molecules Provided with a costimulatory signal, eg, by a stimulated form of a B7 family antigen, eg, by a cell transfected with a nucleic acid encoding a B7 polypeptide and expressing the peptide on its surface, or by a soluble stimulatory form of the peptide obtain. Known cytokines released into the medium can be identified by ELISA or the ability of antibodies to block cytokines and prevent immune cell proliferation or other cell type proliferation induced by cytokines. For example, an IL-4 ELISA kit is available from Genzyme (Cambridge, Mass.) As is the case for IL-7 blocking antibodies. Blocking antibodies against IL-9 and IL-12 are available from Genetics Institute (Cambridge, Mass.). The effect of stimulating or blocking the interaction of B7-4 and PD-1 on the cytokine profile can then be measured.

  The above in vitro immune cell costimulatory assays can also be used in novel cytokine identification methods that can be modulated by modulation of B7-4 and / or PD-1. For example, if stimulation of the CD28 / CTLA4 pathway appears to promote IL-2 secretion, stimulation of the ICOS pathway appears to promote IL-10 secretion (Hutloff et al., 199, Nature 397: 263). If the specific activity induced upon costimulation, for example during immune cell proliferation, cannot be blocked by the addition of blocking antibodies to known cytokines, this activity can result from the action of unknown cytokines. After costimulation, the cytokine is purified from the medium by conventional methods and its activity can be measured by its ability to induce immune cell proliferation.

  In order to identify cytokines that can play a role in the induction of tolerance, the in vitro T cell costimulatory assays described above can be used. In this case, T cells are given a primary activation signal and are contacted with the selected cytokine, but not a costimulatory signal. After washing and resting the immune cells, the cells are re-attacked with both primary activation and costimulatory signals. If immune cells do not respond (eg, proliferate or produce cytokines), they are tolerated and cytokines do not prevent induction of tolerance. However, when immune cells respond, induction of tolerance is blocked by cytokines. Those cytokines that can block the induction of tolerance can be targeted for in vivo blocking with reagents that block B lymphocyte antigens as a more effective means of inducing tolerance in transplant recipients or autoimmune disease subjects. For example, a cytokine blocking antibody can be administered to a subject together with an agent that promotes B7-4 or PD-1 blocking activity.

C. Identification of Molecules that Modulate B7-4 or PD-1 Polypeptide Expression Antibodies produced using the proteins and peptides of the present invention relate to molecules that modulate B7-4 or PD-1 polypeptide expression in cells. Can be used in screening assays. For example, a molecule that modulates an intracellular signaling pathway that culminates in changes in B7-4 or PD-1 polypeptide expression (eg, in response to an activation signal) can be one or more molecules on the cell surface. It can be identified by assaying for expression of a B7-4 or PD-1 polypeptide. Reduction of immunofluorescence staining with the appropriate antibody in the presence of the molecule indicates that the molecule blocks intracellular signaling. Molecules that up-regulate B7-4 or PD-1 polypeptide expression enhance immunofluorescence staining. Alternatively, the effect of a molecule on polypeptide expression can be measured by detecting cellular mRNA levels using the probes of the invention. For example, when a cell expressing a B7-4 or PD-1 polypeptide is contacted with a molecule to be tested, an increase or decrease in mRNA levels in the cell can be determined using standard techniques such as Northern hybridization analysis or a detectable marker. Can be detected by conventional dot blots of mRNA or total poly (A + ) RNA using a cDNA probe labeled with. Molecules that modulate the expression of B7-4 or PD-1 polypeptide are therapeutically useful for up-regulating or down-regulating the immune response alone or in combination with the soluble inhibitory or stimulatory reagents described above. For example, a molecule that inhibits expression of B7-4 can be administered with a second agent, for example, an immunosuppressive agent or a molecule that inhibits expression of PD-1 can be given with an immunostimulant, such as an adjuvant. Examples of molecules that can be tested for the ability to modulate B7-4 or PD-1 include, for example, cytokines such as IL-4, γINF, IL-10, IL-12, GM-CSF and prostaglandins. .

D. Screening assay The present invention binds to modulators, ie B7-4 or PD-1 protein, and has a stimulatory or inhibitory effect on eg B7-4 or PD-1 expression or B7-4 or PD-1 activity Methods for identifying candidates or test compounds or agents (eg, peptides, peptidomimetics, small molecules or other agents) (also referred to herein as “screening assays”) are provided.

  In one aspect, the invention relates to a B7-4 or PD-1 protein or polypeptide or biologically active portion thereof, or which modulates its activity, eg, a B7-4 or PD-1 polypeptide. Assays are provided that screen candidates or test compounds that modulate the ability to interact with an origin binding partner or interactor (interactor) molecule (eg, an intracellular interactor molecule). Test compounds of the present invention are biological libraries, spatially addressable parallel solid or liquid phase libraries, synthetic library methods requiring deconvolution, “one bead one compound” live Can be obtained using any of a variety of methods in combinatorial library methods known in the art, including rally methods and synthetic library methods using affinity chromatography selection. Biological library methods are limited to peptide libraries, and the other four methods are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, KS (1997) Anticancer Drug Des. 12: 145. ).

  Examples of molecular library synthesis methods are described in the art by, for example, DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909, Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91: 11422, Zuckrmann et al. (1994) J. Med. Chem. 37: 2678, Cho et al. (1993) Science 261: 1303, Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2059, Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2061, and Gallop et al. (1994) J. Med. Chem. 37: 1233.

  A library of compounds can be dissolved (eg, Houghten (1992) Biotechniques 13: 412-421), or beads (Lam (1991) Nature 354: 82-84), chips (Fodor (1993) Nature 364: 555-556). Bacteria (Ladner USP 5223409), spores (Ladner USP '409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1865-1869) or phage (Scott and Smith (1990) Science 249: 386) 390), (Devlin (1990) Scence 249: 404-406), (Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6378-6382), (Felici (1991) J. Mol. Biol. 222: 301-310), (Ladner supra).

  In another aspect, the assay expresses a B7-4 target molecule (intracellular interacting molecule or PD-1 receptor) or a PD-1 target molecule (eg, a B7-4 ligand or intracellular interacting molecule). Is a cell-based assay comprising measuring the ability of a test compound to modulate (eg, stimulate or block) the activity of a B7-4 or PD-1 target molecule. The ability of a test compound to modulate the activity of a B7-4 or PD-1 target molecule is determined, for example, when a B7-4 or PD-1 protein binds to or interacts with a B7-4 or PD-1 target molecule Can be accomplished by measuring the ability to do. Measuring the ability of a B7-4 or PD-1 protein to bind to or interact with its binding partner can be done, for example, by measuring direct binding.

In a direct binding assay, the binding of B7-4 or PD-1 protein and B7-4 or PD-1 target molecule can be measured by detecting the labeled protein in the complex. Proteins (or their respective target molecules) can be conjugated with radioisotopes or enzymatic labels. For example, a B7-4 or PD-1 molecule, such as a B7-4 or PD-1 protein, is directly or indirectly labeled with 125 I, 35 S, 14 C or 3 H, and a radioisotope is Can be detected by direct counting or scintillation counting. Alternatively, B7-4 or PD-1 molecules can be enzymatically labeled, for example with horseradish peroxidase, alkaline phosphatase or luciferase, and the enzyme label can be detected by measuring the conversion of the appropriate substrate to product.

  It is also within the scope of this invention to measure the ability of a compound to modulate the interaction between B7-4 or PD-1 and its target molecule without labeling any of the reactants. For example, by using a microphysiometer, the interaction between B7-4 or PD-1 and its target molecule can be detected without labeling either B7-4 or PD-1 or the target molecule ( McConnell, HM et al. (1992) Science 257: 1906-1912). As used herein, a “microphysiometer” (eg, site sensor) is an analytical instrument that measures the rate at which cells acidify their environment using a photoaddressable potentiometric sensor (LAPS). This change in acidification rate can be used as an indicator of the interaction between the compound and the receptor.

  In a preferred embodiment, measuring the ability of a B7-4 or PD-1 protein to bind to or interact with a B7-4 or PD-1 target molecule is determined by measuring the activity of the B7-4, PD-1 or appropriate target molecule. Can be achieved by measuring. For example, the activity of B7-4 or PD-1 or a suitable target molecule can detect the induction of cellular second messengers (eg, tyrosine kinase activity), detect the catalytic / enzymatic activity of a suitable substrate, or reporter Detecting the induction of a gene (including a target responsive regulatory element operably linked to a nucleic acid encoding a detectable marker such as chloramphenicol acetyltransferase) or B7-4, PD-1 Alternatively, it can be measured by detecting a cellular response modulated by an appropriate target molecule. For example, the ability of a B7-4 or PD-1 protein to bind to or interact with a B7-4 or PD-1 target molecule measures the ability of the compound to modulate immune cell costimulation or inhibition, eg, in a proliferation assay. Or by interfering with the ability of the B7-4 or PD-1 polypeptide to bind to an antibody that recognizes a portion of the B7-4 or PD-1 polypeptide.

  Yet another embodiment of the assay of the present invention comprises contacting the B7-4 or PD-1 protein or biologically active portion thereof with a test compound, and the B7-4 or PD-1 protein or biologically active portion thereof of the test compound. It is a cell-free assay that measures the ability to bind to. Binding of the test compound to the B7-4 or PD-1 protein can be measured directly or indirectly as described above. In a preferred embodiment, the assay comprises contacting a known compound that binds to B7-4 or PD-1 to form an assay mixture with a B7-4 or PD-1 protein or biologically active portion thereof, Contacting the test compound and measuring the ability of the test compound to interact with the B7-4 or PD-1 protein, wherein the test compound interacts with the B7-4 or PD-1 protein In determining the ability, the ability of a test compound to bind preferentially to a B7-4 or PD-1 polypeptide or biologically active portion thereof relative to a known compound is measured.

  Another aspect of the assay involves contacting a B7-4 or PD-1 protein or biological portion thereof with a test compound, wherein the test compound modulates the activity of the B7-4 or PD-1 protein or biologically active portion thereof. A cell-free assay that measures the ability to (eg, stimulate or inhibit). Measurement of the ability of a test compound to modulate the activity of a B7-4 or PD-1 protein can be accomplished, for example, by one of the methods described above for measuring direct binding, whereby the B7-4 or PD-1 protein is B7-4 or PD- This can be done by measuring the ability to bind one target molecule. Measurement of the ability of a B7-4 or PD-1 protein to bind to a B7-4 or PD-1 target molecule is also described in real time biomolecular interaction analysis (BIA) (Sjolander, S. and Urbaniczky, C. (1991). Anal.Chem.63: 2338-2345 and Szabo et al. (1995) Curr.Opin.Struct.Biol.5: 699-705). As used herein, “BIA” is a technique for testing biospecific interactions in real time and does not label any of the reactants (eg, BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indicator of real-time reactions between biomolecules.

  In yet another embodiment, the cell-free assay comprises contacting a B7-4 or PD-1 protein or biologically active portion thereof with a known compound that binds to a B7-4 or PD-1 protein to form an assay mixture; Contacting the assay mixture with the test compound and measuring the ability of the test compound to interact with the B7-4 or PD-1 protein, wherein the test compound interacts with the B7-4 or PD-1 protein. Measuring the ability to do includes measuring the ability of a B7-4 or PD-1 protein to bind preferentially to a B7-4 or PD-1 target molecule or modulate its activity.

The cell-free assay of the present invention comprises both soluble and / or membrane bound proteins (eg, B7-4 or PD-1 protein or biologically active portion thereof, or binding partner to which B7-4 or PD-1 binds). This can be done based on the use of the form. For cell-free assays that use membrane-bound forms of the protein (eg, cell surface B7-4 or PD-1 receptor), use solubilizers so that the membrane-bound form of the protein is maintained in solution. May be desirable. Examples of the solubilizer include nonionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, triton ( (Registered trademark) X-100, Triton (registered trademark) X-114, Tesit (registered trademark), isotridecipoly (ethylene glycol ether) n , 3-[(3-colamidopropyl) dimethylaminio] -1-propanesulfonate ( CHAPS), 3-[(3-Colamidopropyl) dimethylaminio] -2-hydroxy-1-propanesulfonate (CHAPSO), or N-dodecyl = N, N-dimethyl-3-ammonio-1-propanesulfonate is there.

  In the above embodiments of the assay method of the present invention, B7-4 or PD-1 or an appropriate target molecule is immobilized to separate a complex-forming form from one or both complex-unformed forms of the protein. It may be desirable to facilitate and adapt to assay automation. Binding of the test compound to the B7-4 or PD-1 protein, or the interaction of the B7-4 or PD-1 protein with the target molecule in the presence and absence of the candidate compound should be suitable to contain the reactants. Any container can be used. Examples of such containers include microtiter plates, test tubes, and microcentrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase / B7-4 or PD-1 fusion protein or glutathione-S-transferase / target fusion protein is adsorbed to glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtiter plates Can then be combined with the test compound or test compound and the non-adsorbed target protein or B7-4 or PD-1 protein and the mixture incubated under conditions that lead to complex formation (eg, physiological conditions with respect to salt and pH) To do. After incubation, the beads or microtiter plate wells are washed to remove any unbound components, and in the case of beads, the matrix is immobilized and the complex measured, for example, directly or indirectly as described above. Alternatively, the complex is dissociated from the matrix, and B7-4 or PD-1 binding or activity levels can be measured using standard techniques.

  Other techniques for immobilizing proteins to the matrix can also be used in the screening assays of the present invention. For example, B7-4 or PD-1 protein or B7-4 or PD-1 target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated B7-4 or PD-1 protein or target molecule is derived from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (eg, biotinylated kit, Pierce Chemical, Rockford, Illinois). It can be manufactured and fixed to the wells of a 96-well plate (Pierce Chemical) coated with streptavidin. Alternatively, antibodies that react with the B7-4 or PD-1 protein or target molecule but do not interfere with the binding of the B7-4 or PD-1 protein to the target molecule are derivatized into the wells of the plate and are The bound target or B7-4 or PD-1 protein can be trapped in the well by antibody conjugation. In addition to the above-described method for GST-immobilized complex, the above-mentioned complex detection method includes a complex immunodetection method using an antibody reactive with B7-4 or PD-1 protein or a target molecule, and B7. There are enzyme binding assays that rely on the detection of enzyme activity associated with the -4 or PD-1 protein or target molecule.

  In another embodiment, measuring the ability of a test compound to modulate the activity of a B7-4 or PD-1 protein is determined by measuring a molecule that functions downstream of B7-4, such as a molecule that interacts with B7-4, or such as PD- This can be done by measuring the ability of a test compound to modulate the activity of a molecule that functions downstream of PD-1 by interacting with one cytoplasmic domain. For example, the level of second messenger can be measured, the activity of an interactor molecule against a suitable target can be measured, or the binding of an interactor to a suitable target can be measured as previously described.

  In another embodiment, a modulator of B7-4 or PD-1 expression is identified by a method of contacting a cell with a candidate compound and measuring the expression of B7-4 or PD-1 mRNA or protein in the cell. The expression level of B7-4 or PD-1 mRNA or protein in the presence of the candidate compound is compared to the expression level of B7-4 or PD-1 mRNA or protein in the absence of the candidate compound. Candidate compounds can then be identified as modulators of B7-4 or PD-1 expression based on this comparison. For example, when the expression of B7-4 or PD-1 mRNA or protein is greater in the presence (eg, statistically significantly greater) than in the absence of the candidate compound, the candidate compound is B7-4 or PD- 1 identified as a stimulator of mRNA or protein expression. Alternatively, a candidate compound is B7-4 or PD when the expression of B7-4 or PD-1 mRNA or protein is less in the presence (eg, statistically significantly less) than in the absence of the candidate compound. Identified as a blocker of -1 mRNA or protein expression. B7-4 or PD-1 mRNA or protein expression levels in cells can be measured by the methods described herein for B7-4 or PD-1 mRNA or protein detection.

  In yet another aspect of the invention, the B7-4 or PD-1 protein, preferably its membrane-bound form, is designated as a “bait protein” in 2-hybrid assays or 3-hybrid assays (see, eg, US Pat. 5283317, Zervos et al. (1993) Cell 72: 223-232, Madura et al. (1993) J. Biol. Chem. 268: 12046-12054, Bartel et al. (1993) Biotechniques 14: 920-924, Iwabuchi et al. (1993) Oncogene 8 : 1693-1696, and Brent, WO 94/10300), other proteins that bind to or interact with B7-4 or PD-1 and are involved in B7-4 or PD-1 activity ("B7-4 or PD -1 binding protein "or" B7-4 or PD-1 bp "). The B7-4 or PD-1 binding protein is also dependent on the B7-4 or PD-1 protein or B7-4 or PD-1 target, for example as an upstream or downstream element of a B7-4 or PD-1 mediated signaling pathway It seems to be involved in signal propagation. Alternatively, the B7-4 or PD-1 binding protein can be a B7-4 or PD-1 inhibitor.

  The two-hybrid system is based on the modular nature of most transcription factors, consisting of separable DNA binding and activation domains. Briefly, this assay uses two different DNA constructs. In one construct, the gene encoding the B7-4 or PD-1 protein is fused to a gene encoding the DNA binding domain of a known transcription factor (eg, GAL-4). In the other construct, a DNA sequence from a DNA sequence library that encodes an unidentified protein (“prey” or “sample”) is fused to a gene encoding an activation domain of a known transcription factor. Is done. When the “bait” and “prey” proteins can interact in vivo to form a B7-4 dependent complex, the DNA binding and activation domains of the transcription factor are in close proximity. This approach allows transcription of a reporter gene (eg, LacZ) operably linked to a transcriptional regulatory site responsive to a transcription factor. Reporter gene expression can be detected and cell colonies containing functional transcription factors can be isolated and used to obtain cloned genes that encode proteins that interact with B7-4 or PD-1 proteins.

  The present invention further relates to a novel drug identified by the above screening assay. Accordingly, it is within the scope of this invention to further use an agent identified according to the description herein in a suitable animal model. For example, an agent identified as described herein (eg, a B7-4 or PD-1 modulating agent, an antisense B7-4 or PD-1 nucleic acid molecule, a B7-4 or PD-1 specific antibody, or B7 -4 or PD-1 binding partner) can be used in animal models to determine the efficacy, toxicity or side effects of treatment with such agents. Alternatively, the mechanism of action of such agents can be determined by using the agents identified according to the description herein in animal models. In addition, the invention relates to the use of novel agents identified by the above screening assays for the treatments described herein.

F. Detection Assays A portion or fragment of the cDNA sequence identified here (and the corresponding complete gene sequence) can be used in various ways as a polynucleotide reagent. For example, using these sequences, (i) each of those genes in the chromosome is mapped, thus identifying the location of the gene region associated with the genetic disease, and (ii) a small biological sample ( Individuals can be identified from (histological type) and (iii) forensic identification of biological samples can be facilitated. These applications are described in the following subsections.

1. Chromosome mapping Once a gene sequence (or part of a sequence) has been isolated, this sequence can be used to map the position of the gene on the chromosome. This process is called chromosome mapping. Thus, a portion or fragment of the B7-4 nucleotide sequence described herein can be used for mapping the location of the B7-4 gene in the chromosome. Mapping the B7-4 sequence to the chromosome is an important first step in clarifying the interrelationship of these sequences with genes associated with disease.

  Briefly, the B7-4 gene can be mapped to the chromosome by producing PCR primers (preferably 15-25 bp in length) from the B7-4 nucleotide sequence. By using computer analysis of the B7-4 sequence, primers can be predicted that do not span multiple exons in the genomic DNA, ie, do not complicate the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the B7-4 sequence will yield an amplified fragment.

  Somatic cell hybrids are produced by fusing somatic cells from different mammals (eg, human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in a random order, but retain mouse chromosomes. Mouse cells cannot grow because they lack a specific enzyme, but human cells retain one human chromosome containing the gene encoding the required enzyme by using a medium that can grow. By using various media, a panel of hybrid cell lines can be established. Since each cell line in a panel contains a single human chromosome or a small number of human chromosomes, and a complete set of mouse chromosomes, mapping of individual genes to specific human chromosomes can be facilitated. (D'Eustachio, P. et al. (1983) Science 220: 919-924). Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.

  PCR mapping of somatic cell hybrids is a rapid method of assigning specific sequences to specific chromosomes. With a single thermal cycler, three or more sequences can be allocated per day. By designing oligonucleotide primers with B7-4 nucleotide sequences, partial location estimation can be achieved with a panel of fragments from specific chromosomes. Similarly, other mapping strategies that can be used to map sequences to their chromosomes include in situ hybridization (Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87: 6223- 27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization with a chromosome-specific cDNA library.

  Furthermore, by using fluorescence in situ hybridization (FISH) of DNA sequences to metaphase chromosome spreads, accurate chromosomal localization can be performed in one step. Chromosome spread can be performed using cells that have been disrupted in metaphase by chemicals that disrupt the mitotic spindle, such as colcemid. Chromosomes can be easily treated with trypsin and then stained with Giemsa. Chromosomes can be identified individually by developing a pattern of light and dark bands on each chromosome. FISH technology can be used with DNA sequences as short as 500 or 600 bases. However, clones larger than 1000 bases are likely to bind to unique chromosomal locations with sufficient signal intensity for simple deletions. Preferably 1000 bases, and more preferably 2000 bases are sufficient to obtain good results in a reasonable amount of time. For a review of this technology, see Verma et al., Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York, 1988).

  Chromosome mapping reagents can be used individually to mark a single chromosome or a single site on that chromosome, or a panel of reagents can be used to mark various sites and / or diverse chromosomes . Reagents corresponding to non-coding regions of the gene are actually preferred for mapping purposes. The coding sequence is likely to be conserved within the gene family, and thus there will be more opportunities for cross-hybridization during chromosome mapping.

  Once the sequence is mapped to the exact chromosomal location, the physical position of the sequence in the chromosome can be correlated with genetic map data (the above data can be found, for example, in McKusick, V., Mendelian Inheritance in Man Available online via the Jones Hopkins University Welch Medical Library). The relationship between genes and diseases, mapped to the same chromosomal region, is then linked, for example as described in Egeland, J. et al. (1987) Nature 325: 783-787 (co-inheritance of physical proximity genes). Can be confirmed through.

  In addition, DNA sequence differences between affected and unaffected individuals with diseases associated with the B7-4 gene can be measured. If a mutation is observed in some or all of the affected individuals, but not at all from unaffected individuals, the mutation is considered to trigger a particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosome, such as deletions or translocations that can be recognized from chromosome spread or detectable using DNA sequence-based PCR. Finally, by completely sequencing genes from several individuals, the presence of the mutation can be confirmed and the mutation from the polymorphism can be distinguished.

2. Tissue typing The B7-4 sequences of the present invention can also be used to identify individuals from a fine biological sample. For example, the United States military is considering the use of a restriction-cut fragment length polymorphism (RFLP) to verify the identity of its personnel. In this technique, a person's genomic DNA is digested with one or more restriction enzymes and probed with a Southern blot to generate a unique band for identification. This method does not imply the limitations of current “recognition tags” that are difficult to positively verify because they can be lost, exchanged or stolen. The sequences of the present invention are useful as additional DNA markers for RFLP (described in US Pat. No. 5,272,057).

  Furthermore, the use of the sequences of the present invention may provide an alternative technique for determining the actual single base DNA sequence of a selected portion from an individual genome. That is, by using the B7-4 nucleotide sequence described herein, two PCR primers can be produced from the 5 ′ and 3 ′ ends of the sequence. These primers can then be used to amplify the individual's DNA followed by its sequencing.

  A panel of corresponding DNA sequences from individuals produced in this way can provide a unique means of individual identification, which has a unique set of DNA sequences because each individual has a different allele. It depends. By using the sequences of the present invention, the above identified sequences can be obtained from individuals and tissues. The B7-4 nucleotide sequence of the present invention represents a unique portion of the human genome. Allelic variation occurs to some extent in the coding regions of these sequences and to a significant extent in non-coding regions. It has been estimated that allelic variation between individuals occurs with a frequency of about once per 500 bases. Each of the sequences described herein can be used as a standard to which, to some extent, DNA from individuals can be compared for identification purposes. Due to the large number of polymorphisms found in non-coding regions, fewer sequences are required to identify individuals. The non-coding sequence of SEQ ID NO: 1 or 3 can comfortably provide a positive means of individual identification with a panel of perhaps 10-1000 primers each resulting in a non-coding amplified sequence of 100 bases. If the predicted coding sequence is used, a more appropriate primer number for positive individual identification would be 500-2000.

  When creating an individual specific identification database by using a panel of reagents from the B7-4 nucleotide sequences described herein, those same reagents can later be used for tissue identification from the individual. With a unique identification database, a positive means of confirming living or dying individuals can be created from very small tissue samples.

3. Use of Partial B7-4 Sequences in Forensic Biology DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field that uses genotyping of biological evidence found in crime scenes, for example, as a means of positively identifying criminal offenders. To make such confirmation, DNA sequences collected from very small biological samples found in crime scenes, such as tissues, such as hair or skin, or body fluids such as blood, saliva or semen, by using PCR techniques Can be amplified. The amplified sequence can then be compared to a standard, thereby confirming the source of the biological sample.

  By using the sequences of the present invention, for example, by providing another “identification marker” (ie, another DNA sequence that is unique to a particular individual), the reliability of DNA-based forensic identification means can be increased. Polynucleotide reagents targeted to specific locations in the human genome, such as PCR primers, can be provided. As stated above, the actual base sequence information can be used for identification as an accurate alternative to the pattern formed by the restriction enzyme generated fragments. Sequences targeted to the non-coding region are particularly suitable for this application because the large number of polymorphisms found in the non-coding region makes it easier to identify individuals using this technique. Examples of polynucleotide reagents include B7-4 nucleotide sequences or portions thereof having a length of at least 20 bases, preferably at least 30 bases.

  In addition, by using the B7-4 nucleotide sequences described herein, polynucleotide reagents, such as labels or labels that can be used to identify specific tissues, such as brain tissue, in situ hybridization techniques. Possible probes can be provided. This is very useful when a forensic pathologist is presented with an unknown source of tissue. The panel of B7-4 probes can be used for tissue confirmation by species and / or organ type.

  In a similar manner, these reagents, such as B7-4 primers or probes, can be used for screening tissue cultures for contaminants (ie, screening for the presence of a mixture of different types of cells in the culture).

G. Predictive Medicine The present invention also relates to the field of predictive medicine in which individuals are treated prophylactically by using diagnostic assays, predictive assays and monitoring clinical trials for predictive (predictive) purposes. Accordingly, one aspect of the present invention provides B7-4 or PD-1 protein and / or nucleic acid expression and B7-4 or PD-1 activity in the context of biological samples (eg, blood, serum, cells, tissues). Relates to a diagnostic assay to measure whether the individual suffers from or is at risk of developing a disease or disorder associated with abnormal forms of B7-4 or PD-1 expression or activity No is determined. The present invention also provides a prognostic (or predictive) assay for determining whether an individual is at risk for developing a disease associated with B7-4 or PD-1 protein, nucleic acid expression or activity. For example, mutations in the B7-4 or PD-1 gene can be assayed in a biological sample. By using the above assay for prognostic or predictive purposes, individuals can be treated prophylactically prior to the onset of diseases characterized by or related to B7-4 or PD-1 protein, nucleic acid expression or activity. Assays described herein, such as the diagnostic assay or the assay described below, can also be used to detect the tendency to spontaneous abortion.

Another aspect of the invention relates to monitoring the effects of agents (eg, drugs, compounds) on B7-4 or PD-1 expression or activity in clinical trials.
These and other drugs are described in more detail in the sections below.

  1. Diagnostic assays

  In an example of a method for detecting the presence or absence of B7-4 or PD-1 protein or nucleic acid in a biological sample, the biological sample is obtained from a test subject and the B7-4 or PD-1 protein or nucleic acid is detected. A compound or agent capable of detecting a B7-4 or PD-1 protein or a nucleic acid encoding a B7-4 or PD-1 protein (eg, mRNA, genomic DNA) such that its presence is detected from a biological sample Contact the biological sample. A preferred agent for detecting B7-4 or PD-1 mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to B7-4 or PD-1 mRNA or genomic DNA. The nucleic acid probe is, for example, a human B7-4 or PD-1 nucleic acid, such as the nucleic acid of SEQ ID NO: 1, 3, 10 or 11 or a portion thereof, such as an oligonucleotide at least 15, 30, 50, 100, 250 or 500 nucleotides in length. Yes, and may be sufficient to specifically hybridize with B7-4 or PD-1 mRNA or genomic DNA under stringent conditions. Other suitable probes for use in the diagnostic assays of the invention are also described herein.

A preferred B7-4 or PD-1 protein detection agent is an antibody capable of binding to B7-4 or PD-1 protein, preferably an antibody with a detectable label. The antibody may be polyclonal or preferably monoclonal. An intact antibody or a fragment thereof (eg, Fab or F (ab ′) 2 ) can be used. The term “labeled” with respect to a probe or antibody refers to direct labeling of the probe or antibody by binding (ie, physically binding) a detectable substance to the probe or antibody, as well as another reagent that is directly labeled. Indirect labeling of a probe or antibody due to the reactivity of Examples of indirect labeling include a method in which a primary antibody is detected using a fluorescently labeled secondary antibody and a method that can be detected with fluorescently labeled streptavidin by end-labeling a DNA probe with biotin. The term “biological sample” is intended to encompass tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, by using the detection method of the present invention, B7-4 or PD-1 mRNA, protein, or genomic DNA in a biological sample can be detected in vitro and in vivo. For example, in vitro detection techniques for B7-4 or PD-1 mRNA include Northern hybridization and in situ hybridization. In vitro detection techniques for B7-4 or PD-1 protein include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation and immunofluorescence. Southern hybridization is a technique for in vitro detection of B7-4 or PD-1 genomic DNA. Further, in vivo detection techniques for B7-4 or PD-1 protein include techniques for introducing a labeled anti-B7-4 or PD-1 antibody into a subject. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard techniques.

  In one embodiment, the biological sample contains protein molecules from the test compound. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a serum sample isolated from a subject by conventional means.

  In another embodiment, the method of the present invention further obtains a control biological sample from a control subject so that the presence of B7-4 or PD-1 protein, mRNA or genomic DNA is detected from the biological sample, Contacting a control sample with a compound or agent capable of detecting B7-4 or PD-1 protein, mRNA or genomic DNA, and the presence of B7-4 or PD-1 protein, mRNA or genomic DNA in the control sample in the test sample Compare with the presence of B7-4 or PD-1 protein, mRNA or genomic DNA.

  The invention also includes a kit for detecting the presence of B7-4 or PD-1 in a biological sample. For example, the kit can be a labeled compound or agent capable of detecting B7-4 or PD-1 protein or mRNA in a biological sample, a means for measuring the amount of B7-4 or PD-1 in the sample, and Means can be included for comparing the amount of B7-4 or PD-1 to a standard. The compound or agent can be packaged in a suitable container. The kit may further include instructions for using the kit to detect B7-4 or PD-1 protein or nucleic acid.

2. Prognostic Assays Further, by using the diagnostic methods described herein, subjects at risk for developing a disease or disorder associated with abnormal B7-4 or PD-1 expression or activity can be recognized. For example, the assays described herein, such as the aforementioned diagnostic assays or the assays described below, can be used to recognize subjects at risk of developing a disease associated with B7-4 or PD-1 protein, expression or activity. Can be used. That is, the present invention relates to abnormal B7-4 or PD-1 expression or activity in which a test sample is obtained from a subject and B7-4 or PD-1 protein or nucleic acid (eg, mRNA, genomic DNA) is detected. A method for identifying a disease or disorder, wherein the presence of B7-4 or PD-1 protein or nucleic acid is suffering from or is associated with a disease or disorder associated with abnormal B7-4 or PD-1 expression or activity Provide a method that is characteristic of the subject at risk. As used herein, “test sample” includes a biological sample obtained from a subject of interest. For example, the test sample can be a biological fluid (eg, serum), a cell sample, or a tissue.

  In addition, using the prognostic assay described herein, abnormalities can be caused by administering a drug (eg, agonist, antagonist, peptide mimetic, protein, peptide, nucleic acid, small molecule or other drug candidate) to a subject. It can be determined whether a disease or disorder associated with type B7-4 or PD-1 expression or activity can be treated. That is, the present invention is a method for measuring whether a subject can be effectively treated with a drug for diseases associated with abnormal B7-4 or PD-1 expression or activity, obtaining a test sample, Methods for detecting B7-4 or PD-1 protein or nucleic acid expression or activity (eg, abundance of B7-4 or PD-1 protein or nucleic acid expression or activity is caused by administration of a drug to form abnormal B7-4 Or a disease associated with PD-1 expression or activity that is characteristic for a subject that can be treated).

  In addition, by using the method of the present invention, is it possible to detect a genetic alteration in the B7-4 or PD-1 gene so that the subject whose gene is altered is at risk of developing a disease associated with the B7-4 or PD-1 gene? No can be determined. In a preferred embodiment, the method comprises at least one modification that affects the integrity of a gene encoding a B7-4 or PD-1 protein or misexpression of a B7-4 or PD-1 gene in a cell sample from a subject. Detecting the presence or absence of a genetic modification characterized by one. For example, the genetic modification may include: 1) deletion of one or more nucleotides from the B7-4 or PD-1 gene, 2) one or more nucleotides into the B7-4 or PD-1 gene Addition, 3) Substitution of one or more nucleotides of B7-4 or PD-1 gene, 4) Chromosomal translocation of B7-4 or PD-1 gene, 5) Messenger RNA of B7-4 or PD-1 gene Alteration of transcript level, 6) aberrant modification of B7-4 or PD-1 gene, eg in the case of methylation pattern of genomic DNA Presence of type splicing pattern, 8) non-wild type level of B7-4 or PD-1 protein, 9) allelic loss of B7-4 or PD-1 gene, Beauty 10) B7-4 or PD-1 of the inappropriate post translational protein modifications may be detected by checking at least one of presence. As described herein, a number of assay techniques are known in the art that can be used to detect alterations in the B7-4 or PD-1 gene. A preferred biological sample is a tissue or serum sample isolated from a subject by conventional means, such as a heart tissue sample.

  In certain embodiments, detection of the modification is performed by polymerase chain reaction (PCR) (see, eg, US Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR or alternatively, ligation chain reaction (LCR) (eg, Landegran (1988) Science 241: 1077-1080, and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91: 360-364), the latter requires B7 -4 or PD-1 gene may be particularly useful for detecting point mutations (see Abravaya et al. (1995) Nucleic Acids Res. 23: 675-682). This method involves taking a sample of cells from a patient, isolating nucleic acid (eg, genome, mRNA or both) from the sample cells, and allowing hybridization and amplification of the B7-4 or PD-1 gene (if present). Contacting the nucleic acid sample with one or more primers that specifically hybridize with the B7-4 or PD-1 gene under the conditions performed and detecting the presence or absence of the amplification product or Detecting the size and comparing the length with a control sample can be included. It is anticipated that PCR and / or LCR may be desirable to use as a preliminary amplification step with any of the techniques used for mutation detection described herein.

  Alternative amplification methods include continuous sequence replication (Guatelli, JC et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcription amplification system (Kwoh, DY et al. (1989) Proc. Natl Acad. Sci. USA 86: 1173-1177), Q-beta replicase (Lizardi, PM et al. (1988) Biotechnology 6: 1197), or other nucleic acid amplification methods, followed by techniques well known to those skilled in the art. The molecule amplified using the detection is performed. These detection schemes are particularly useful for the detection of such molecules when the number of nucleic acid molecules present is very low.

  In another embodiment, mutations in the B7-4 or PD-1 gene from sample cells can be confirmed by modification of the restriction enzyme cleavage pattern. For example, sample and control DNA is isolated, amplified (if desired), digested with one or more restriction endonucleases, and fragment length sizes are measured by gel electrophoresis and compared. A difference in fragment length size between sample and control DNA indicates a mutation in the sample DNA. Furthermore, the use of sequence specific ribozymes (see, eg, US Pat. No. 5,498,531) can be used to assess the presence of specific mutations due to the development or loss of ribozyme cleavage sites.

  In other embodiments, genetic mutations in B7-4 or PD-1 are identified by hybridizing sample and control nucleic acids, such as DNA or RNA, to a high density array containing hundreds or thousands of oligonucleotide probes. (Cronin, MT et al. (1996) Hum. Mutat. 7: 244-255, Kozal, MJ et al. (1996) Nat. Med. 2: 753-759). For example, genetic mutations in B7-4 or PD-1 can be confirmed in a two-dimensional array containing light-generated DNA probes as described in Cronin, MT et al. (1996) supra. . Briefly, a first hybridization array of probes can be used to confirm base changes between sequences by scanning long extended strands of DNA in samples and controls and creating a linear array of continuous overlapping probes. . This stage confirms the point mutation. After this stage, the specific hybridization is characterized by using a small specialized probe array that is complementary to all the mutations or mutations detected by the second hybridization array. Each mutation array is composed of parallel probe sets, one complementary to the wild type gene and the other complementary to the mutant gene.

  In yet another aspect, direct sequencing of the B7-4 or PD-1 gene is performed by using any of a variety of sequencing reactions known in the art, and the sequence of sample B7-4 or PD-1 Mutations can be detected by comparing to the corresponding wild type (control) sequence. Examples of sequencing reactions were developed by Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74: 560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74: 5463). There is something based on technology. It also includes sequencing by mass spectrometry (eg, PCT International Publication No. WO 94/16101, Cohen et al. (1996) Adv. Chromatogr. 36: 127-162, and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38: 147-159), any of a variety of automated sequencing methods could be utilized when performing a diagnostic assay ((1995) Biotechniques 19: 448).

  Other methods of detecting mutations in the B7-4 or PD-1 gene include detecting mismatched bases in RNA / RNA or RNA / DNA heteroduplexes using protection from cleaving agents (Myers (1985) Science 230: 1242). In general, “mismatch cleavage” techniques in the art involve hybridizing a potential mutant RNA or DNA obtained from a tissue sample with a (labeled) RNA or DNA containing a wild type B7-4 or PD-1 sequence. By providing a heteroduplex formed by Double stranded duplexes are treated with agents that cleave the single stranded region of the duplex, eg, the region present due to base pair mismatches between the control and sample strands. For example, the mismatch region can be enzymatically digested by treating the RNA / DNA duplex with ribonuclease and treating the DNA / DNA hybrid with S1 nuclease. In other embodiments, the mismatch region can be digested by treating the DNA / DNA or RNA / DNA duplex with hydroxylamine or osmium tetroxide and piperidine. After digestion of the mismatch region, the resulting material is separated by size on a denaturing polyacrylamide gel and the mutation site is measured. See, eg, Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85: 4397, Saleeba et al. (1992) Methods Enzymol. 217: 286-295. In preferred embodiments, control DNA or RNA can be labeled for detection.

  In yet another embodiment, the mismatch cleavage reaction is a mismatch base pair in double stranded DNA in a system identified to detect and map point mutations in B7-4 or PD-1 cDNA obtained from a sample of cells. One or more proteins that recognizes (so-called “DNA mismatch repair” enzymes) are used. For example, the E. coli mutY enzyme cleaves the G / A mismatch A, and the thymidine DNA glycosylase from HeLa cells cleaves the G / T mismatch T (Hsu et al. (1994) Carcinogenesis 15: 1657-1662). According to an illustrative embodiment, a probe based on a B7-4 sequence, such as a wild type B7-4 or PD-1 sequence, is hybridized with a cDNA or DNA product from the test cell (s). If the duplex is treated with a DNA mismatch repair enzyme and there are cleavage products, they can be detected such as from electrophoresis protocols. See, for example, US Pat. No. 5,459,039.

  In other embodiments, mutations in the B7-4 or PD-1 gene can be confirmed by utilizing changes in electrophoretic mobility. For example, by using single strand conformation polymorphism (SSCP), differences in electrophoretic mobility between mutant and wild type nucleic acids can be detected (Orita et al. (1989) Proc. Natl. Acad. Sci. USA 86: 2766, Cotton (1993) Mutat. Res. 285: 125-144, and Hayashi (1992) Genet. Anal. Tech. Appl. 9: 73-79). Single-stranded DNA fragments of sample and control B7-4 or PD-1 nucleic acids can be denatured and renatured. The secondary structure of a single-stranded nucleic acid changes depending on the sequence, and even a single base change can be detected by changing the electrophoretic mobility. The DNA fragment can be labeled or detected with a labeled probe. The sensitivity of the assay can be increased by using RNA (rather than DNA), in which case the secondary structure is more sensitive to sequence changes. In a preferred embodiment, the subject method separates double-stranded heteroduplex molecules based on changes in electrophoretic mobility by using heteroduplex analysis (Keen et al. (1991) Trends Genet. 7: 5).

  In yet another embodiment, denaturing gradient gel electrophoresis (DGGE) is used to assay the movement of mutant or wild-type fragments in polyacrylamide gels containing denaturing agent gradients (Myers et al. (1985) Nature 313: 495). . When DGGE is used as an analytical method, it can be ensured that the DNA is not completely denatured by adding modifications to the DNA, for example by adding a GC clamp of high melting point GC-rich DNA of about 40 bp by PCR. In other embodiments, temperature gradients are used instead of denaturing gradients to confirm differences in mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys. Chem. 265: 12753).

  Examples of other point mutation detection techniques include, but are not limited to, selective oligonucleotide hybridization, selective amplification or selective primer extension. For example, oligonucleotide primers can be made that hybridize with target DNA under conditions that allow hybridization only if a known mutation is centrally located and then a perfect match is found (Saiki et al. (1986) Nature 324: 163, Saiki (1989) Proc. Natl. Acad. Sci. USA 86: 6230). When the oligonucleotide is bound to the hybridization membrane and hybridized with the labeled target DNA, the allele-specific oligonucleotide is hybridized with the PCR amplified target DNA or some different mutants.

  Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in connection with the present invention. Oligonucleotides used as primers for specific amplification can be of interest in the center of the mutation (hence amplification by subtractive hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17: 2437-2448). Alternatively, it can be carried at the tip 3 ′ end of one primer (Prossner et al. (1993) Tibtech 11: 238) where polymerase extension can be blocked or reduced by mismatch under appropriate conditions. Furthermore, it may be desirable to create a detection means based on cleavage by introducing a new restriction site into the mutated region (Gasparini et al. (1992) Mol. Cell Probes 6: 1). In certain embodiments, it is expected that amplification can also be accomplished using amplification Taq ligase (Barany (1991) Proc. Natl. Acad. Sci. USA 88: 189). In such a case, the ligation reaction occurs only when there is a perfect match at the 3 'end of the 5' sequence, so that the presence of a known mutation at a specific site can be detected by looking for the presence or absence of amplification Become.

  The methods described herein can be performed, for example, by utilizing a prepackaged diagnostic kit comprising at least one probe nucleic acid or antibody reagent as described herein, conveniently for example clinically By using in a setting, patients presenting with signs or family history of a disease or illness involving the B7-4 or PD-1 gene can be diagnosed.

  Furthermore, any cell type or tissue in which B7-4 or PD-1 is expressed can be utilized in the prognostic assay described herein.

VII. Administration of B7-4 or PD-1 Modulating Agent The B7-4 or PD-1 modulating agent of the invention is directed to a subject in a biologically compatible form suitable for in vivo pharmaceutical administration to promote or suppress immune cell-mediated immune responses. Be administered. “Biocompatible forms suitable for in vivo administration” include the case where the therapeutic effect of the protein is more influential than the toxic effect in the form of the protein being administered. The term subject matter is intended to encompass living organisms in which an immune response can be induced, eg, mammals. Examples of subjects include humans, dogs, cats, mice, rats and transgenic species thereof. Administration of the agents described herein can be performed in a pharmacological form comprising a therapeutically active amount of the agent alone or in combination with a pharmaceutically acceptable carrier.

  Administration of a therapeutically active amount of the therapeutic composition of the present invention is defined as an amount effective for the dosage and duration necessary to achieve the desired result. For example, the therapeutically active amount of a B7-4 or PD-1 polypeptide can vary depending on various factors, such as the individual's condition, age, sex and weight, and the ability of the peptide to induce a desired response in the individual. By adjusting the dosing regimen, an optimal therapeutic response can be provided. For example, several divided doses can be administered daily or the dose can be reduced proportionally as indicated by the urgency of the treatment situation.

  B7-4 or PD-1 modulators (eg, peptides, nucleic acid molecules, antibodies, peptidomimetics or small molecules) can be administered in a conventional manner, eg injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application. Or it can be administered by rectal administration. Depending on the route of administration, the active compound may be coated with substances that protect the compound from the action of enzymes, acids and other natural conditions that can inactivate the compound. For example, in order to administer a B7-4 or PD-1 modulating agent by a method other than parenteral administration, the peptide is coated with a substance that prevents inactivation of the peptide, or the substance and the peptide are co-administered. It may be desirable.

  The B7-4 or PD-1 modulating agent can be administered to the individual in a suitable carrier, diluent or adjuvant, and can be co-administered with the enzyme inhibitor or in a suitable carrier such as a liposome. Pharmaceutically acceptable diluents include saline and aqueous buffer. Adjuvant is used in its broadest sense and includes immunostimulatory compounds such as interferons. Possible adjuvants here include resorcinols, nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DEEP) and trasilol. Liposomes include water-in-oil-in-water emulsions and conventional liposomes (Sterna et al. (1984) J. Neuroimmunol. 7:27).

  The active compound may also be administered parenterally or intraperitoneally. Dispersions can also be made in glycerin, liquid polyethylene glycols and mixtures thereof and oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.

  Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Long-term absorption of the injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

  A sterile injectable solution is required after containing the active compound (eg, B7-4 or PD-1 polypeptide) in the required amount in a suitable solvent with one or a combination of the ingredients listed above. And can be produced by sterile filtration. Generally, dispersions are prepared by including the active compound in a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the production of sterile injectable solutions, the preferred production methods are vacuum drying and lyophilization, if there are active ingredients (eg peptides) and additional desired ingredients from the previously sterile filtered solution A powder containing it is also produced.

  In the manner described above, if the active compound is suitably protected, the protein can be administered orally, for example, with an inert diluent or an assimilable dietary carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as conventional media or agents are incompatible with the active compounds, their use in therapeutic compositions is contemplated. Supplementary active compounds can also be included in the compositions.

  It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. The unit dose form used herein includes physically independent units suitable as unit doses for the mammalian subject to be treated, each unit producing the desired therapeutic effect with the required pharmaceutical carrier. A predetermined amount of active compound calculated as follows. Specifics regarding the unit dosage forms of the present invention include: (a) the characteristics specific to the active compound and the specific therapeutic effect to be achieved; and (b) the field in formulating the active compound to treat sensitivity in an individual. Directly defined by inherent limitations.

  In one aspect of the invention, a therapeutically effective amount of an antibody against B7-4 or PD-1 protein is administered to a subject. The therapeutically effective amount (ie, effective dose) of an antibody as defined herein is about 0.001 to 30 mg / kg (body weight), preferably about 0.01 to 25 mg / kg (body weight), more preferably about 0.00. It is in the range of 1 to 20 mg / kg (body weight), more preferably about 1 to 10 mg / kg, 2 to 9 mg / kg, 3 to 8 mg / kg, 4 to 7 mg / kg or 5 to 6 mg / kg (body weight). One of ordinary skill in the art can influence the doses required to effectively treat a subject, including the severity of the disease or disorder, prior treatment, the subject's general health and / or It should be appreciated that age, as well as other illnesses, may be included but not limited. Moreover, treatment of a subject with a therapeutically effective amount of an antibody can include a single treatment or, preferably, can include a series of treatments. In preferred examples, the subject is about 1 to 10 weeks, preferably about 2 to 8 weeks, more preferably about 3 to 7 weeks, more preferably about 4, 5 or 6 weeks, about 0.1 to about 1 weekly. Treat with antibody in the range of 20 mg / kg body weight. It is also contemplated that the effective dose of antibody used for treatment may increase or decrease over the course of a particular treatment. The change in dose may be due to the results of the diagnostic assays described herein.

  Monitoring the effects of drugs (eg, drugs or compounds) on B7-4 or PD-1 protein expression or activity can be applied in both basic drug screening and clinical trials. For example, the effectiveness of a drug as measured by the screening assays described herein as increasing B7-4 or PD-1 gene expression, protein levels or upregulating B7-4 or PD-1 activity is: It can be monitored in clinical trials of subjects exhibiting B7-4 or PD-1 gene expression, decreased protein levels, or downregulation of B7-4 or PD-1 activity. On the other hand, the effectiveness of a drug measured by a screening assay as reducing B7-4 or PD-1 gene expression, protein levels, or down-regulating B7-4 or PD-1 activity is B7-4 or It can be monitored in clinical trials of subjects exhibiting PD-1 gene expression, increased protein levels, or upregulation of B7-4 or PD-1 activity. In the above clinical trials, the expression or activity of the B7-4 or PD-1 gene, and preferably other genes involved in the disease, is used as a “read out” or marker of specific cell phenotypes. obtain.

  While not intended to be limiting, for example, with an agent (eg, compound, drug or small molecule) that modulates B7-4 or PD-1 activity (eg, as identified in a screening assay described herein) Genes that are modulated in cells by treatment of, for example B7-4 or PD-1, can be identified. Thus, for example, to test the effect of a drug on a B7-4 or PD-1 related disease in a clinical trial, cells are isolated, RNA is prepared, B7-4 or PD-1 and B7-4 or PD-1 Each can be analyzed for the expression level of other genes involved in related diseases. The gene expression level (ie, gene expression pattern) is determined by the Northern blot analysis or RT-PCR described herein, or alternatively, by one of the methods described herein. It can be quantified by measuring the amount or by measuring the activity level of B7-4 or PD-1 or other genes. In this regard, gene expression patterns can serve as markers, which are indicators of cellular physiological responses to drugs. This response state can thus be measured at various time points before and during drug treatment of the individual.

  In a preferred embodiment, the present invention provides for the treatment of a subject with an agent (eg, an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule or other drug candidate identified by a screening assay described herein). A method for monitoring efficacy, comprising: (i) obtaining a pre-dose sample from a subject prior to drug administration, and (ii) determining the expression level of B7-4 or PD-1 protein, mRNA or genomic DNA in the pre-dose sample. Detecting, (iii) obtaining one or more post-administration samples from the subject, (iv) detecting the expression or activity level of B7-4 or PD-1 protein, mRNA or genomic DNA in the post-administration samples; (V) Expression or activity level of B7-4 or PD-1 protein, mRNA or genomic DNA in the pre-dose sample And (vi) thereby modifying the administration of the drug to the subject, wherein the method comprises a step of comparing the administration of the drug to the B7-4 or PD-1 protein, mRNA or genomic DNA in the sample (s) after administration. provide. For example, increased drug administration may be a desirable means to increase B7-4 or PD-1 expression or activity above a detected level, ie, increase the effectiveness of the drug. On the other hand, reducing drug administration may be a desirable means to reduce B7-4 or PD-1 expression or activity below the detected level, ie, reduce the effectiveness of the drug. According to such embodiments, B7-4 or PD-1 expression or activity can be used as an indicator of drug efficacy, even in the absence of an observable phenotypic response.

  The invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as drawings and sequence listings, are hereby incorporated by reference.

(Example)
Example 1. Isolation of B7-4 cDNA Molecules Public databases were searched for nucleic acid molecules encoding homologous polypeptides by using the protein sequence of the extracellular domain of human B7-1. Two overlapping sequences AA292201 and AA399416 in the EST database were identified. Using these sequences, full-length B7-4 cDNA was isolated from human activated keratinocytes and placental cDNA libraries as follows.

From these ESTs, oligonucleotides having the sequence 5′-CAGCTATGGTGGGCCGCACTACAA-3 ′ (SEQ ID NO: 5) and 5′-AGGTGCTAGGGGACAGTGTTAGACA-3 ′ (SEQ ID NO: 6) were synthesized. By using these oligonucleotides, as a template, cDNA prepared by reverse transcription of mRNA from spleen, activated B cells, INF-γ activated keratinocytes, normal spleen and placenta in follicular lymphoma cases PCR reaction was primed. The conditions were 94 ° C. for 1 minute, 94 ° C. for 30 seconds, 56 ° C. for 30 seconds, 68 ° C. for 1 minute 35 cycles; 68 ° C. for 3 minutes and 4 ° C. hold. All templates gave a predicted size band of 389 bp. The 389 bp product from PCR of INF-γ activated keratinocytes was purified by agarose gel electrophoresis and 0.12 ng was used as a template in a PCR reaction containing 0.05 mmol of biotin-21-dUTP and the above primers. The conditions were 94 ° C. for 1 minute, 94 ° C. for 30 seconds, 56 ° C. for 30 seconds, 68 ° C. for 2 minutes, 20 cycles; 68 ° C. for 5 minutes, 4 ° C. hold. The biotinylated PCR product was purified with a nucleospin column (Clontech) and used as a probe in the Clone Capture cDNA sorting method (Clontech). 60 ng of denatured biotinylated PCR product was incubated with 2 mM CoCl 2 , 1 × RecA buffer, 1 μg RecA protein, 1 × ATP in a final volume of 30 μl. The reaction was incubated at 37 ° C. for 15 minutes. To the mixture, 0.7 μg of activated keratinocyte cDNA library plasmid DNA and 0.4 μg of human placenta cDNA library were added and incubation continued for 20 minutes. 50 ng of EcoRV digested lambda DNA was added to the reaction and incubated for 5 minutes. 0.6 μl of 10% SDS and 5.6 μg of proteinase K were added and incubated at 37 ° C. for 10 minutes. Proteinase K was inactivated by adding 1 μg of 0.1 molar PMSF. Streptavidin magnetic beads are preincubated with 5 μg of salmon sperm DNA sheared for 10 minutes, the beads are captured with a magnet, the supernatant is removed, and the beads are washed with 30 μl of binding buffer (1 mmol EDTA, 1 mol NaCl, 10 mmol). In Tris-HCl, pH 7.5). The beads were added to the reaction and the reaction was incubated for 30 minutes with gentle mixing at room temperature. The beads were captured with a magnet and the supernatant was removed. The beads were washed with 1 ml wash buffer (1 mmol EDTA, 2 mol NaCl, 10 mmol Tris-HCl, pH 7.5), the beads were captured with a magnet and the supernatant was removed. The washing procedure was repeated 3 times. 1 ml of sterile H 2 O was added to the washed beads and incubated for 5 minutes at 37 ° C., the beads were captured with a magnet and the supernatant was removed. Elute the captured DNA by adding 0.1 ml elution buffer (1 mmol EDTA, 0.1 N NaOH) and incubating at room temperature for 5 minutes, capture the beads with a magnet, and remove the supernatant. Saved in a new tube. 22.5 μl of precipitation mixture containing carrier and pH neutralizer was added along with 2.5 volumes of ethanol. Plasmid DNA was concentrated by centrifugation and redissolved in H 2 O. Plasmid DNA was reintroduced into E. coli DH10B / P3 by electroporation and selected on LB-agar plates containing 7.5 μg / ml tetracycline and 25 μg / ml ampicillin. The colonies are picked up on a nytran filter and the sequences of 5′-CAGCTATGGTGGGCCGAACTACAA-3 ′ (SEQ ID NO: 7), 5′-AGGTGCTAGGGGACAGTGTTAGCACA-3 ′ (SEQ ID NO: 8) and 5′-TCGCCTGTTAGTCGCCACCACCATA-3 ′ (SEQ ID NO: 9) Hybridized with 32 P-labeled oligonucleotide with All oligos are from the AA292201 sequence. The final wash conditions were 2 × SSC, 0.1% SDS, 55 ° C. for 20 minutes. Two hybridizing colonies were picked and the sequence of the cDNA insert was determined.

  Sequencing showed two forms of B7-4 molecule. The first form of secreted B7-4 (B7-4S) encodes a protein with a short hydrophobic domain without a membrane anchor. This form of nucleotide and amino acid sequence is shown in SEQ ID NOs: 1 and 2, respectively. The second form of B7-4 membrane (B7-4M) encodes a protein with a transmembrane and short cytoplasmic domain. This form of nucleotide and amino acid sequence is shown in SEQ ID NOs: 3 and 4, respectively. Both members of the identified B7-4 family have signal, IgV and IgC domains, as illustrated in FIGS. The B7-4M form was calculated using the default Blosum62 matrix with gap penalties set at presence 11 and extension 1 under conditions where B7-1 and B7-2 have about 26% identity (http : //www.ncbi.nlm.nih.gov) It has about 21% amino acid identity with human B7-1 and about 20% amino acid identity with human B7-2.

Example 2 B7-4 mRNA expression: Northern blot analysis The soluble form of B7-4 mRNA is expected to be about 1.2 kb, although other sizes are possible. The second form of mRNA is approximately 3.8 kb, and a few mRNAs are 1.6 and 6.5 kb.

B7-4 polypeptide expression was analyzed. RNA was prepared by homogenization with guanidine thiocyanate and cesium chloride centrifugation. An equal amount of RNA (approximately 2 μg poly (A) + RNA) is subjected to agarose gel electrophoresis, blotted, and hybridized with a portion of the 32 P-labeled B7-4 cDNA common to both B7-4S and B7-4M forms. It was. These B7-4 mRNAs are highly expressed in the placenta, lung and heart and moderately expressed in the thymus. In addition, these B7-4 mRNAs are weakly expressed in skeletal muscle, kidney, pancreas, prostate, testis, ovary, small intestine, colon and peripheral blood leukocytes. They have also been found to be very weakly expressed in the liver or brain. B7-4 mRNA was not expressed in unstimulated monocytes but was strongly induced by IFN-γ. Similarly, expression of these polypeptides was found to be induced in keratinocytes by TPA / IFN-γ and in dendritic cells by IFN-γ. These B7-4 mRNAs were not expressed in unstimulated B cells, but were induced by Ig crosslinking.

  In addition, the expression of these B7-4 mRNAs was examined in various cell lines. They were not found to be expressed in B cell lines such as Raji, Ramos, LBL, Nalm6 and DHL-4. They were also not expressed in T cell lines such as Jurkat, Rex, CEM, HPB-ALL, Peer4 and H9 or HTLV-1 transformed T cell lines such as SPP and MT2 or spinal line U937.

Example 3 Further Characterization of B7-4 mRNA Expression: Northern Blot Analysis Mouse and human multiple tissue Northern blots (Clontech, Palo Alto, Calif.) Were analyzed at 32 P in Quik Hyb (Stratagene, La Jolla, Calif.) According to manufacturer's instructions. Probed with dCTP radiolabeled cDNA probe. The human B7-4 probe was composed of a 1 kb BamHI / NotI fragment of cDNA spanning the coding region of SEQ ID NO: 1 and the 3 ′ untranslated region. The mouse B7-4 probe was composed of a 300 bp cDNA fragment from the coding region. Control actin probe was supplied by Clontech. Blots were washed twice in 2 × SSC, 0.1% SDS at room temperature, then in 0.2 × SSC, 0.1% SDS at 65 ° C. and examined by autoradiography.

  B7-4 mRNA was expressed at high levels in heart, human placenta and human fetal liver, and at low levels in spleen, lymph nodes, thymus and mouse liver.

  B7-4 mRNA is expressed in various traits including PU5-1.8, RAW264.7, K-Balb, M-MSV-Balb / 3T3, Hepa1-6, R1.1, L1210, P38D1, P815 and NB41A3 cells. Expressed in transformed mouse cell lines.

Example 4 Further characterization of B7-4 mRNA expression: quantitative PCR, gene chip hybridization, and RNA blot analysis B7-4 mRNA expression in antigen presenting cells was examined and compared to the expression of B7-1 and B7-2 in those cells did. For quantitative PCR analysis, cellular RNA was treated with deoxyribonuclease, re-extracted, and converted to first strand cDNA. FAM (6-carboxyfluorescein) -labeled human B7-4, B7-1, B7-2 and GAPDH probes were purchased from PE Biosystems (B7-4: Primer 5′-GCCGAAGTCATCTTGGACAAG-3 ′ (SEQ ID NO: 13) and 5′-TCTCAGTGGCTGGTCACAT-3 ′ (SEQ ID NO: 14), probe 5′-FAM-CACCACCACCAATTCCAAGA-3 ′ (SEQ ID NO: 15), B7-1: primer 5′-ACGTGAACCAAGGAAGTGAAGAAA-3 ′ (SEQ ID NO: 16) and 5′- TGCCAGCTCTTCAACAGAAACAT-3 ′ (SEQ ID NO: 17), probe 5′-FAM-TGGCAACGCTGTCCTGTGTCAC-3 ′ (SEQ ID NO: 18), B7-2: primer 5′-GG GCCGCAAAAGTTTTGAT-3 ′ (SEQ ID NO: 19) and 5′-GCCCTTGTCCCTTGATCTGAAGA-3 ′ (SEQ ID NO: 20), probe 5′-FAM-CGGACAGTTGGACCCTGAGACTTCACA-3 ′ (SEQ ID NO: 21)).

  PCR reactions were set up in 96-well plates using reagents from the Perkin Elmer TaqMan ™ EZ kit according to the manufacturer's instructions. A standard curve was constructed for each of the 4 genes analyzed. Forty cycles of PCR were performed on an ABI prism 7700 sequence detector and the results for B7-4, B7-1 and B7-2 were normalized using GAPDH.

  Affymetrix Mu19KsubA chip was used for gene chip hybridization analysis. The sequence of a portion of murine B7-4 is represented by the expressed sequence tag TC17781 of The Institute for Genomic Research on this chip. RNA isolation, chip hybridization and scanning were performed as described in Byrne, M.C. et al. (2000) Curr.Prot.Mol.Biol.Suupl.49: 22.2.1-22.2.13.

For RNA blot hybridization, 1.6 kb human and 3.6 kb murine B7-4 cDNA was excised by XbaI digestion and labeled by random priming with Klenow fragment of γ- 32 P-ATP and DNA polymerase I. RNA blots were hybridized as described by Freeman, GJ et al. (1992) J. Immunol. 149: 3795-3801.

Human dendritic cells were removed from peripheral blood. Mononuclear cells were isolated after fractionation on a Ficoll gradient. Non-adherent cells were removed and the remaining cells were cultured in 150 ng / ml human GM-CSF (R & D Systems) and 100 ng / ml human IL-4 (R & D Systems) for 2 days. Non-adherent dendritic cells were isolated (CD80 + CD86 + HLA-DR + CD54 + CD58 + CD1a + ) and cultured with GM-CSF alone or GM-CSF, 2.5 μg / ml LPS (Sigma Chemicals) and Activation by 10 μg / ml human interferon-γ. Cells were harvested 4 and 20 hours after activation and RNA was isolated using the RNeasy kit (Qiagen).

Granulocytes, lymphocytes and Ia + cells were immunodepleted from murine bone marrow mononuclear cells by magnetic activated cell sorting and cultured with GM-CSF and IL-4 in Petri dishes. When dendritic cells were harvested as a non-adherent population after 7 days in culture, they were demonstrated to be 75-80% CD11c + , high IA + cells. Cells were activated with LPS and human interferon-γ.

  Analysis of expression in human blood mononuclear cells by RNA blot hybridization demonstrated that B7-2 is not expressed by unstimulated mononuclear cells, but is rapidly upregulated upon interferon-γ treatment. Treatment of mononuclear cells with another pro-inflammatory cytokine, tumor necrosis factor (TNF) -α, resulted in a low level of induction similar to that found with medium alone, but this led to plastic It seems to be the result of activation by adhesion. In addition to the major 4.2 kb B7-4 mRNA, a minor group of 1.8 kb B7-4 mRNA was also observed in interferon-γ treated mononuclear cells. B7-4 expression was also observed by human B cells activated by cell surface immunoglobulin cross-linking, but not by the Raji cell line. Similarly, B7-1 is not expressed by unstimulated mononuclear cells, but is upregulated in response to interferon-γ with kinetics similar to B7-4 expression. In contrast, B7-2 mRNA is constitutively expressed in mononuclear cells and interferon-γ or TNF-α treatment does not affect levels.

  In addition, B7-4, B7-1 and B7-2 mRNA expression by human dendritic cells was examined by quantitative PCR. Dendritic cells derived from human peripheral blood were treated with granulocyte-macrophage colony stimulating factor (GM-CSF) alone or activated with GM-CSF, lipopolysaccharide (LPS) and interferon-γ. As a result of activation with LPS and interferon-γ, B7-4 mRNA was rapidly induced in non-induced cells with a 16-fold increase after 4 hours and a 34-fold increase after 20 hours. B7-1 and B7-2 mRNA were also induced after activation. B7-1 was induced 21-fold after 4 hours and 22-fold after 20 hours. B7-2 showed little induction after 4 hours, but expression was induced 5-fold after 20 hours. The expression of B7-4 by dendritic cells from murine bone marrow treated with LPS and interferon-γ was examined using GeneChip ™ hybridization. B7-4 expression in these cells follows a pattern similar to that observed in human dendritic cells, with a 5-fold induction of B7-4 mRNA relative to uninduced cells at 6 and 20 hours after induction. It was done. These data indicate that B7-4 is expressed by antigen presenting cells and lymphocytes and that it is induced in dendritic cells in a manner similar to B7-1 and B7-2. Treatment of human keratinocytes with phorbol ester and interferon-γ also induced B7-4.

  In murine tissues, an approximately 3.7 kb B7-4 mRNA transcript was detected by Northern blot hybridization. The distribution of murine B7-4 mRNA closely approximated that of human B7-4, with high levels in heart, thymus and lung, and low levels in kidney, spleen and liver.

Example 5 FIG. Chromosomal localization of B7-4 Chromosomal localization of the B7-4 gene was measured using a single chromosome blot kit available from Quantum (Toronto, Canada). The blot was probed with a sequence that recognizes both B7-4S and B7-4M. Using this method, B7-4 polypeptide was localized to human chromosome 9, and B7-1 and B7-2 were localized to human chromosome 3. Butyrophilins also shared limited amino acid sequence identity with the B7-4 group and were localized to the major histocompatibility gene complex on chromosome 6. The chromosomal location of B7-4 was confirmed using B7-4 specific primers in PCR amplification of a monochromosomal somatic hybrid DNA template available from Quantum Technologies (Canada).

Example 6 Binding of B7-4 molecules to T cell ligands or antibodies COS cells were transfected with vector DNA (pcDNAI) or expression plasmids containing B7-4M cDNA. After 72 hours, transfected COS cells were detached by incubation in PBS containing 0.5 mM EDTA for 30 minutes at 37 ° C.

  The ability of COS cells expressing B7-4M to bind to various T cell receptors and antibodies was tested. FACS analysis of binding of CD28Ig, CTLA4-Ig and control Ig by B7-4 transfected COS cells showed that neither CD28Ig nor CTLA4-Ig binds to B7-4 (FIG. 8). The ability of COS cells expressing B7-4M to bind to IgG and murine ICOS-his fusion protein was also tested. No binding between human B7-4 and murine ICOS was detected (FIG. 9). As shown in FIG. 10, FACS analysis showed binding of BB1 (anti-B7-1 and anti-B7-3), but IgM or 133 (anti-B7) antibody against B7-4 transfected COS cells. Was not shown.

Example 7 Costimulation of T cell proliferation by B7-4 molecules The ability of B7-4 polypeptides to costimulate human T cell proliferation was tested.
As previously reported (Gimmi, CD et al. (1993) Proc. Natl. Acad. Sci. USA 90, 6586-6590), monoclonal antibodies directed against B cells, natural killer cells and macrophages were used. Human CD28 + T cells were isolated by immunomagnetic bead depletion. B7-4 and vector transfected COS cells were harvested 72 hours after transfection, incubated with 25 μl / ml mitomycin-C for 1 hour and washed thoroughly. 10 5 T cells that have not been experimented were stimulated with 20000 mitomycin-c treated COS cells transfected with plate-bound anti-CD3 mAb + the indicated DNA construct.

  T cell proliferation was measured by 3H-thymidine (1 μCi) incorporated in the last 12 hours of the 72 hour incubation. As shown in FIGS. 11 and 12, COS cells expressing B7-4 can costimulate T cell proliferation.

Example 8 FIG. Generation of murine antibodies against B7-4 A mammalian expression vector (pEF6 or pcDNA3.1 (Invitrogen)) containing the entire murine or human B7-4 cDNA was prepared. The cDNA / vector construct was dissolved in 0.9% saline at 1 mg / ml (not TE or PBS).

  Prior to immunization, 78 μl of 1 mg / ml cardiac toxin (Sigma # C-1777) in 0.9% saline was injected into the anterior tibial muscle of each hind leg of the immunized mouse. Each mouse was then left alone for 5 days.

  After anesthetizing the mice, 50 μl of 1 mg / ml purified B7-4 cDNA / vector construct (0.9% in saline) was injected into each pre-regenerative tibial muscle.

  Antibody titers were measured approximately 6 days after immunization using standard methods, for example, by ELISA assay. cDNA immunization was repeated every 2-4 weeks for 3 cycles (until antibody titers were> 1: 10000). The mice were then boosted with CHO cells transfected with PDL-1.

  Spleen cells isolated from mice with appropriate antibody titers were collected. Hybridomas were produced by fusing spleen cells with the fusion partner SP2-0). Hybridomas and antibodies were engineered using standard methods (see, eg, “Antibodies: A Laboratory Manual”, Harlow, E. and Lane, D., Cold Spring Harbor Laboratory (1988), incorporated herein by reference. Some).

  Antibodies 2A3, 10D9, 5A9 and 11D12 were included in those selected in the screening assay. These antibodies were found to bind to COS or CHO cells transfected with human B7-4 but not to mock transfected cells or cells transfected with mouse B7-4. By using antibodies, the presence of B7-4 in various cell populations was detected. B7-4 expression was observed particularly in heart tissue, tumor cells (including lung tumor cells, ovarian tumor cells, breast tumor cells, epithelial tumor cells and squamous cell carcinoma), placenta and thymic epithelium.

Example 9 Production of fully human antibodies to B7-4 In this example, fully human antibodies to B7-4 or PD-1 are produced in mice that are transgenic for human immunoglobulin genes. Are transgenic mice produced using standard methods, for example according to Hogan et al., “Manipulating the Mouse Embryo: A Laboratory Manual” (Cold Spring Harbor Laboratory, incorporated herein by reference). Or purchased. Published methods of embryonic stem cells (Teratocarcinomas and embryonic stem cells: a practical approach, edited by Robertson, EJ, IRL Press, Washington DC, 1987, Zjilstra et al. (1989) Nature 342: 435-438, and Schwartzberg et al. (1989) Science 246: 799-803, each of which is incorporated herein by reference). The DNA cloning method is described in Sambrook, J. et al., Molecular Cloning: A Laboratoty Manual, Second Edition (1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, incorporated herein by reference). It is carried out according to Oligonucleotides are synthesized or purchased, for example, on an Applied Biosystems oligonucleotide synthesizer according to specifications provided by the manufacturer.

  Transgenic mice are immunized with a fusion protein comprising at least an immunogenic portion of purified or recombinant B7-4 or PD-1 or B7-4 or PD-1 extracellular domain. Each mouse is injected intraperitoneally with approximately 400 μg of B7-4 or PD-1 in 100 μL of phosphate buffered saline (PBS). Approximately 6 days later, serum samples are collected by retroorbital sinus bleeding.

  Antibody reactivity and specificity for B7-4 or PD-1 is assessed using an indirect enzyme linked immunosorbent assay (ELISA). The antibody specificity of the antibody for B7-4 or PD-1 is analyzed by testing several immunoglobulin superfamily molecules as controls (eg, CTLA4 and CD28). Antibodies with human variable regions that bind to B7-4 or PD-1 are detected by enzyme conjugates specific for human IgM and human IgG subclasses that have no cross-reactivity with mouse immunoglobulins. Briefly, PVC microtiter plates are coated with B7-4 or PD-1 by coating the wells overnight at 37 ° C. with 5 μg / mL B7-4 in PBS. Serum samples are diluted in PBS, 5% serum, 0.5% Tween-20, 1 hour at room temperature in the wells, then anti-human IgG Fc and IgG F (ab ′) horseradish peroxidase or anti-human in the same dilution Incubate with IgM Fc horseradish peroxidase. After 1 hour at room temperature, enzyme activity is assessed by adding ABTS substrate (Sigma, St. Louis, MO) and read after 30 minutes at 415-490 nm. Pre-immune serum samples from the same mice are also tested for human antibody titers against the same target antigen.

  Spleen cells isolated from mice with appropriate antibody titers are harvested. Hybridomas are produced by fusing spleen cells to a suitable fusion partner (eg, myeloma cells). Manipulate hybridomas and antibodies according to “Antibodies: A Laboratory Manual” Ed Harlow and David Lane (Cold Spring Harbor Laboratory (1988), hereby incorporated by reference).

  Humanized antibodies to B7-4 or PD-1 can be generated by grafting into the framework regions of human immunoglobulin using the murine VH and VL domain complementarity determining sequences of the murine antibody (Riechmann et al., 1988). Nature 332: 323, Verhoeyen et al., 1988, Science 239: 1534).

Example 10 Generation of human single-chain Fv reactive with B7-4 or PD-1 As an alternative method for producing monoclonal antibody-secreting hybridomas, anti-B7-4 or anti-PD-1 antibody (single chain Fv-like portion of the antibody) Were identified and isolated by screening a combinatorial library of human immunoglobulin sequences represented by the M13 bacteriophage from Melbourne, Cambridge Antibody Technology, Limited (Winter et al., 1994, Annu. Rev. Immunol. 1994, 12: 433, Hoogenboom et al., 1998, Immunotechnology 4: 1). By using PD-1.Fc or B7-4.Fc, immunoglobulin library members that bind to B7-4 or PD-1 polypeptide were isolated. Phage display library generation and screening kits are commercially available and scFvs were generated using standard methods (Helfrich et al., J. Immunol. Methods 2000, 237: 131-45, Cardoso et al., Scand. J. Immunol 2000, 51: 337-44). PD-1.Fc or B7-4.Fc was immobilized on plastic and phage expression specific scFvs were selected by panning and multi-round enrichment (Griffiths et al., 1993, EMBO J., 12: 725).

Example 11 Identification of receptors for B7-4 Human PD-1 extracellular region fused to the hinge CH2-CH3 domain of human immunoglobulin gamma 1 or murine Ig gamma 2a (with mutations that block FcR and complement interactions) ) Was used to search for a ligand that binds to PD-1. As part of this search, staining of the cell surface of monocytes stimulated with gamma-interferon was found. As observed by Northern blot hybridization, B7-4 is induced in monocytes following stimulation with gamma-interferon.

  Binding of PD-1-Fc (human Ig gamma 1) to the surface of COS cells transiently transfected with the B7-4 expression vector was tested. COS cells were transfected with B7-4M or B7-1 using LipofectAMINE transfection reagent. After 48 hours, cells were stained with anti-IgG conjugated to human PD-1-Fc, murine PD-1-Fc, CTLA4-Fc, Flt4-Fc or IgG followed by phycoerythrin (PE). The cells were then analyzed by flow cytometry. As shown in FIG. 13, COS cells expressing B7-4 bound to both human PD-1-Fc and murine PD-1-Fc, but with CTLA4-Fc, Flt4-Fc or human IgG Did not combine. As a positive control, it was demonstrated that B7-1 expressing COS cells bind CTLA4-Fc but not PD-1-Fc or IgG.

  In addition, in situ assays of transfected COS cell monolayers were performed. Monolayers were probed with PD-1Fc, CTLA4Fc or human IgG1, and binding was detected with a secondary antibody directed against the Fc moiety and conjugated to alkaline phosphatase. Binding was visualized by the chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium and light microscopy. In parallel, cells transfected with B7-4 bind to PD-1-Fc, but CTLA4-Fc (human Ig gamma 1) or Flt4-Fc, murine Flt4 bound to human Ig gamma 1 It was found that it does not bind to the extracellular region of. In parallel, PD-1Fc did not bind to the surface of mock transfected COS cells, the surface of COS cells transfected with B7-1 or B7-2.

  In another experiment, using a BIACORE-based assay, no binding of PD-1-Fc to the soluble form of B7-1 or B7-2 was detected and binding to B7-4 was detected. In parallel, hCTLA4 was shown to bind to B7-1 but not B7-4. PD-1-Fc or CTLA4-Fc was immobilized and the conditions were essentially the same as described by Fitz et al. (1997) Oncogene 15: 613. Concentrated COS cell medium from cells transfected with full length B7-4M or B7-4-Fc was injected and real-time biomolecular interaction analysis (BIA) (Sjolander, S. and Urbaniczky, C. (1991) Anal Chem. 63: 2338-2345 and Szabo et al. (1995) Curr.Opin.Struct.Biol. 5: 699-705). Human B7-4 was found to bind to human and mouse PD-1, and this binding was blocked by competition with PD-1-Fc rather than co-injected CTLA4-Fc. These experiments demonstrate the binding of soluble B7-4-Fc fusion protein to immobilized PD-1-Fc and the presence of a soluble form of B7-4 in the conditioned medium of B7-4M cDNA transfected cells. This is probably the result of running down.

  FIG. 14 illustrates the ability of PD-1 but not Flt4 (the receptor for vascular endothelial growth factor C) to competitively inhibit PD-1 binding to B7-4. The binding of human PD-1 gamma 2a fusion protein to COS cells expressing B7-4M is shown in panel A. This binding was detected by an anti-gamma 2a specific reagent conjugated to PE. Human PD-1 bound to IgG1 was added at 50 μg / ml, 6.25 μg / ml, 100 μg / ml or 25 μg / ml and was found to compete for binding. As a control, Flt4IgG1 at 100 μg / ml was not found to compete for PD-1 binding to B7-4.

  In yet another experiment, the ability of B7-4 to bind to PD-1 was measured by flow cytometry and BIACORE binding assay. As detected by flow cytometry (FIG. 15), human and murine PD-1.Ig fusion proteins bound to both human and murine B7-4 expressed in CHO cells. However, neither human CTLA-4.Ig, human CD28.Ig, or human ICOS.Ig bound to the B7-4 expressing cell line. The PD-1 fusion protein did not bind to CHO cells transfected with the vector alone. Further confirmation of the PD-1: B7-4 interaction was achieved using surface plasmon resonance with a BIACORE instrument. Human and murine PD-1.Ig protein and human CTLA-4.Ig were immobilized on the flow cell surface of dextran chips and tested for binding to soluble human B7-4.Ig. B7-4.Ig bound to both human and murine PD-1.Ig but not to human CTLA-4.Ig (FIG. 16). This binding was blocked by competition with co-injected soluble PD-1.Ig, but not in the case of CTLA-4.Ig. The soluble forms of human B7-1 and B7-2 did not bind to immobilized human PD-1.

  These data demonstrate that PD-1 binds to B7-4 and that this interaction can modulate the action of PD-1.

Example 12 FIG. B7-4 can transmit negative signals to immune cells.
In this example, 5 × 10 5 Jurkat T cells per well were stimulated with anti-CD3 coated beads (1: 1 ratio) and soluble anti-CD28. Cells expressing B7-4 or a negative control, so-called EZZ, were titrated into the wells. Supernatants were collected at 48 hours and assayed by ELISA for human IL-2. FIG. 17 shows that IL-2 production decreases with increasing number of COS B7-4 cells (right bar in the figure).

  Using a similar assay format, for example, when human PHA blasts from PBMC were stimulated with immobilized anti-CD3 and soluble anti-CD28, a decrease in T cell proliferation was observed by titration with COS cells expressing B7-4. It was done.

Example 13 The PD-1: B7-4 interaction blocks CD3-mediated T cell proliferation.
In order to examine the functional significance of the PD-1: B7-4 interaction, the functional consequences of the interaction between B7-4 and its receptor were also examined using human T cells. Peripheral blood mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation. The CD4 + T cell population (85-90% purity) was purified by a negative selection method using a cocktail of monoclonal antibodies and immunomagnetic beads (Perceptive Biosystems). Anti-CD3, control IgG and fusion protein were polyurethane coated tosyl activated dynabeads as previously reported (Blair, P. et al. (1998) J. Immunol. 160: 12-15) according to manufacturer's instructions. (Dinal) was covalently bonded. The indicated concentration of anti-CD3 antibody (UCHT1, Pharmingen) was added to 1 × 10 7 beads / ml (0.1 molar phosphate buffer, pH 7.4). Control IgG was added to the bead suspension to maintain a constant total Ig concentration of 5 μg / ml during binding. Similarly, anti-CD3 / B7-4.Ig (γ2a) beads are prepared at the indicated anti-CD3 antibody concentration, where B7-4.Ig accounts for 40% of total bound protein (2 μg / 10 7 beads). The control IgG was at a constant concentration that made up all of the remaining total binding protein. 10 5 T cells were cultured in 96-well flat bottom plates and beads were added at the ratio of 1 bead to 1 cell in the presence or absence of the indicated concentrations of anti-CD28 antibody (CD28.2, Pharmingen). Proliferation was measured by labeling the last 6 hour culture of the 4-day assay with 1 μCi 3 H thymidine / well. For analysis by cytokine ELISA, cultures were set up as above and supernatants were collected at the indicated times. Interferon-γ, IL-10 and IL-2 concentrations were measured using a commercially available ELISA kit (Genzyme).

Purified CD4 + T cells obtained from peripheral blood mononuclear cells (PBMC) were activated with beads coated with anti-CD3 mAb and human B7-4.Ig or control Ig. Proliferation and cytokine production was assessed 96 hours after stimulation. As shown in FIG. 18, cells activated with anti-CD3 mAb / B7-4.Ig coated beads showed a 69% reduction in proliferation relative to anti-CD3 mAb / control Ig activated cells. Furthermore, cytokine activation was also reduced by cell activation in the presence of B7-4. In the presence of B7-4, interferon-γ and IL-10 secretion decreased at rates of about 80% and 60%, respectively (FIG. 18). IL-2 production failed to be detected under these activation conditions both after 24 and 96 hours. However, under conditions co-stimulated with a soluble anti-CD28 form, cell activation in the presence of B7-4 reduced IL-2 production. That is, activation of murine and human T cells in the presence of B7-4 prevents both proliferation and cytokine secretion.

Example 14 The result of the PD-1: B7-4 interaction depends on the intensity of the T cell receptor and the CD28 signal.
To examine the relationship between T cell receptor, CD28 and PD-1 signaling, human CD4 + with a fixed concentration of B7-4.Ig and an increasing concentration of soluble anti-CD28 mAB with suboptimal or optimal concentration of anti-CD3 mAB. T cells were stimulated. By using anti-CD3 mAB coated beads, the sub-optimal limit and the concentration required for optimal T cell stimulation were established. Under suboptimal T cell receptor binding conditions (1 μg / ml anti-CD3 mAb), a minimal increase was observed in the absence of costimulation (FIG. 19A). Addition of increasing concentrations of soluble anti-CD28 mAB increased proliferation by a factor of 30. Under these conditions, T cell activation in the presence of B7-4 reduced proliferation by 80% (FIG. 19A). A maximum level of costimulation (250 ng / ml anti-Cd28) was required to regain the inhibition of proliferation transmitted by B7-4 stimulation. In contrast, under saturated conditions of T cell receptor activation (2 μg / ml anti-CD3 mAB), B7-4 transmission inhibition of T cell proliferation was observed only in the absence of CD28 costimulation (FIG. 19B).

Example 15. Ability of B7-4 to block CD28 signal and cytokine production Since the inhibitory effect of the PD-1: B7-4 pathway appears to be determined by the intensity of the signal through TCR and CD28 (see previous examples), Weak CD3 / CD28 transduction responses are easily down-regulated. To test the interaction of the CD28 signal and PD-1: B7-4 pathway, pre-activated DO11.10 CD4 + T cells were treated with CHO-IA d /B7.2 or CHO-IA d /B7.2/B7−. It was activated by the OVA peptide presented by 4.

To detect B7-4, 5 × 10 4 CHO transfectants were incubated with 5 μg / ml human PD-1Ig (hPD-1-Ig) (Genetics Institute, Cambridge, Mass.) And goat anti-murine Developed with IgG2a-Phycoerythrin (PE) (Southern Biotechnology Associates, Inc., Alabama). In addition, cells were stained separately with 5 μg / ml anti-IA-PE or B7.2-PE (Pharmingen, San Diego, Calif.). After each stage, the cells were washed 3 times with PBS / 1% BSA / 0.02% sodium azide. After the final incubation, the cells were fixed with 1% paraformaldehyde. 10,000 events were analyzed with a FACS Caliber (Becton Dickinson, Mountain View, CA). All isotype controls were all derived from Farmingen.

Spleen cells were prepared from DO11.10 mice by treatment with tris -NH 4 Cl, remove red blood cells. Cells were treated with 10FCS (Sigma, St. Louis, MO), 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 250 ng / ml amphotericin B, 10 mM HEPES, 50 micromolar 2-ME. (All from Life Technologies) and 1 mg / ml OVA peptide (analyse) for 72 hours in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 15 mg / ml gentamicin (BioWitacker, Walkersville, MD) Tikal Biotechnology Services, Boston, Massachusetts). When CD4 + T cells were purified by a positive selection method using a magnetically activated cell sorting column (Miltenyi Biotech, Auburn, Calif.), The purity was> 98%. Cells were rested overnight before restimulation.

Incubation with 50 μg / ml mitomycin C (Bristol Laboratories, Princeton, NJ) for 16 hours at 37 ° C. prevented the growth of CHO cells. At the end of the incubation period, cells were harvested with 10 mM EDTA in PBS, washed twice and left on ice for 1 hour. Subsequently, the cells were washed 3 times and resuspended in culture medium. 10 5 pre-activated CD4 + T cells were cultured with various concentrations of OVA peptide and 10 4 mitomycin C-treated CHO transfectants in 96 well plates. To assay for proliferation, the cultures were incubated for 48 hours and pulsed with 1 μCi / well of [ 3 H] thymidine (New England Nukelia, Boston, Mass.) For the final 6 hours of the incubation period.

B7 and IA d expression was similar in all CHO transfectants (FIG. 20). As expected, the introduction of B7.2 increased the proliferative response by T cells at all antigen concentrations (FIG. 21). However, B7-4 blocked the response at low peptide concentrations (0.01 μg / ml and 0.001 μg / ml) (FIG. 21).

To examine the ability of the PD-1: B7-4 pathway to block cytokine production, supernatants from DO11.10CD4 + T cells activated by OVA peptides presented by CHO cell transfectants were analyzed. Aliquots of the supernatant were collected at various times after the start of culture. IL-2, IL-4, IFN-γ and IL-10 levels were analyzed using recombinant cytokine standards from mAbs and Pharmingen. The detection limits were as follows: IL-2, 20 pg / ml, IL-4, 40 pg / ml, IFN-γ, 100 pg / ml and IL-10, 200 pg / ml. When DO11.10 CD4 + T cells were cultured with 0.1 μg / ml peptide and B7-4, production of IL-2, IL-4, IFN-γ and IL-10 was markedly blocked (FIG. 22). . At this concentration there was only a weak growth inhibition. However, B7-4 markedly inhibited cytokine production with 0.01 μg / ml peptide, consistent with growth inhibition (FIG. 23). IL-10 was not detected under these activation conditions. Thus, PD-1 ligation by B7-4 can down-regulate cytokine production even when T cell proliferation is not affected.

A ribonuclease protection assay was used to determine if the decrease in cytokine production was due to a decrease in mRNA levels. CD4 + T cells were restimulated with various CHO cell transfectants and 0.01 μg / ml OVA peptide. After 48 hours, cells were harvested and mRNA was isolated using TRIzol ™ reagent (Life Technologies). 5 μg of mRNA was analyzed for cytokine levels by ribonuclease protection assay using RiboQuant multiprobe kit mCK1 (Pharmingen) according to manufacturer's instructions. After stimulation with 0.01 μg / ml of OVA peptide presented by CHO-IA d /B7.2, IL-4, IL-10, IL-13, IL-2, in pre-activated DO11-10CD4 + T cells Transcript levels of IL-6 and IFN-γ mRNA were detected. However, the introduction of B7-4 significantly reduced cytokine mRNA levels. In unstimulated T cell cultures or T cells activated by the presented peptides by CHO-IA d, upregulation of mRNA for cytokines was negligible. Furthermore, these results indicate that the PD-1: B7-4 pathway has the ability to antagonize strong B7 / CD28 signals at least when antigenic stimuli are weak or limited, and strong antigenic stimuli It proves that at least cytokine production is blocked under conditions.

Example 16 Mechanism of Action of the PD-1: B7-4 Pathway Cross-linking of CTLA-4 has been shown to block cell cycle progression in T cells that have not undergone specific experiments (Krummel. MF and Allison, JP). (1996) J. Exp. Med. 183: 2533-2540, Walunas, TL et al. (1996) J. Exp. Med. 183: 2541-2550). Since PD-1 was isolated from a murine cell line undergoing apoptosis, a possible mechanism of action of the PD-1: B7-4 pathway could increase programmed cell death. To address this issue, DO11.10 CD4 + T cells were restimulated with 0.01 μg / ml peptide and various CHO transfectants and analyzed for cell cycle progression. CD4 + T cells were restimulated with 0.01 μg / ml peptide as before. After 36 hours of culture, cells were harvested and stained with anti-CD4-FITC. Cells were washed with PBS, fixed in 70% ethanol on ice for 1 hour, and then resuspended in PBS containing 10 μg / ml ribonuclease (Sigma) and 50 μg / ml propidium iodide (Sigma). Analysis was performed within 1 hour of staining.

After 48 hours, cells were harvested and stained with CD4-FITC. After permeabilization, the cells were incubated with propidium iodide and analyzed for G 0 / G 1 , S / G 2 and subdiploid populations. CD4 + T cells restimulated with the peptide presented by CHO-IA d have the majority of cells in the subdiploid population that are indicative of apoptosis (FIG. 24). In cultures in which CD4 + T cells were stimulated with peptides presented by CHO-IA d / B7-2, the number of cells in the S / G 2 phase increased and the number in the subdiploid population decreased. It was found that the cells were in their cycle and were rescued from apoptosis by B7 / CD28 costimulation. The introduction of B7-4 increased the number of cells in the G0 / G1 phase (FIG. 24). B7-4 cultures had a level of apoptosis comparable to that of CHO-IA d / B7 cultures. This was confirmed by annexin staining. Blocking cell progression by the PD-1: B7-4 pathway confirms its role in down-regulation of T cell activation.

Example 17. Inhibition of Biotinylated Human B7-4Fc Binding to Human PD-1Fc Fc fusion proteins were generated by binding the extracellular region of PD-1 or B7-4 to the hinge-CH2-CH3 domain of murine Igγ2a. Recombinant proteins were produced in CHO cells transiently transfected with Lipofectamine (Gibco-BRL) or stable transfected CHO cell lines and purified from conditioned media using protein A-Sepharose.

  The ability of antibodies against B7-4 or PD-1 to block human B7-4Fc and human PD-1Fc interaction was tested using standard ELISA methods. Briefly, human PD-1 Fc molecules were immobilized on 96 well plates, blocked and washed. Biotinylated B7-4Fc molecule (100 ng / ml) was added to the wells at concentrations of about 2000, 700, 200, 70, 25, 8, and 1.18 ng / ml (FIG. 25). Wells were incubated with horseradish peroxidase conjugated with StrepAvidin, washed and developed using standard methods. The ED50 of B7-4Fc was found to be 108 ng / ml.

  Murine antibodies against human B7-4 (10D9 and 11D12) or the scFv portion of human immunoglobulin (B7-4-1, B7-4-6 and B7-4-12) are biotinylated human B7 to human PD-1Fc. The ability to block -4Fc binding was tested with seven concentrations of inhibitor. IC50 was found to be in the range of 0.5 nanomolar to 24 nanomolar and the data is shown in FIG.

Also, using the same ELISA method as described above, PD-1 specific scFvs were tested for their ability to block B7-4Fc binding to PD-1Fc. Human scFv reactive with PD-1 (PD1-17 scFv) was found to block specific binding as shown in FIG. 26 (EC50 between 10 −7 and 10 −8 ). Complete IgG was generated using the VL and VH domains of PD1-17scFv. Briefly, VH and VL coding regions were linked to the genomic CH and CL gene sequences of the expression vector. The resulting expression vector was transiently transfected into human 293 cells and IgG was harvested from the conditioned medium. The potency of all transplanted IgG molecules was higher than that of scFv antibody (EC50 between 10-8 and 10-9 moles).

Example 18 Administration of soluble B7-4Fc exacerbates the disease in a murine model.
Experiments that can share many clinical and pathological features with the human disease multiple sclerosis to determine whether modulation of the B7-4 / PD-1 pathway has immunomodulatory activity in vivo Protein was evaluated in a murine model of experimental autoimmune encephalomyelitis (EAE). Female SJL / J mice were immunized with 100 μg of proteolipid protein (PLP) in complete Freund's adjuvant. Ten days later, the spleen was removed and prepared into a single cell suspension and then restimulated in vitro with 5 μg PLP for 96 hours. Cells were washed three times in PBS and then 15 × 10 6 cells were transferred to SJL / J mice that had not undergone a specific experiment by intraperitoneal injection. Adoptive transfer of autoreactive T cells induces acute paralysis in the recipient mouse, which manifests itself as loss of normal sensitivity of the tail, followed by complete hind leg paralysis. And proceed. This onset of paralysis is consistent with a marked infiltration of activated T cells and macrophages in the CNS. Under most conditions, this is an acute model of illness with spontaneous recovery after a short period of paralysis. To evaluate B7-4Fc, mice were injected subcutaneously (n = 10) with 200 μg of 100 μl of sterile saline in 0, 2, 4, 7 and 11 days after cell transfer. Control mice (n = 10) received only an equal volume of saline. All animals were regularly monitored for clinical signs of disease and evaluated as follows: Loss of normal sensitivity of the tail, 2. 2. Poor hind leg / partial hind leg paralysis; 3. Complete hind leg paralysis; 4. Rear and front leg paralysis, Drowned state.

  In the experiment shown in FIG. 27, the development and onset of clinical disease was similar in both groups. However, mice treated with B7-4Fc developed severe disease and the majority of animals progressed rapidly to complete and forelimb paralysis (9/10 and 1/10 for B7-4Fc and control mice, respectively). ). Mortality related to clinical signs of illness was 10% in the control group and 70% in B7-4Fc treated mice. Furthermore, despite the fact that the treatment was stopped on day 11, the recovery of clinical pathology was substantially delayed in B7-4Fc treated mice that were actually alive.

  In conclusion, using an autoimmune adoptive transfer model mediated by T cells, administration of soluble B7-4Fc results in increased clinical signs of disease, resulting in increased mortality and recovery from paralysis Is late. These findings are consistent with the high activation / invasion of inflammatory cells into the CNS, clearly demonstrating the immunomodulatory potential for the B7-4Fc protein in vivo.

Equivalent Content Those skilled in the art will recognize many equivalents for the embodiments of the invention described herein, or they should be able to ascertain them using only routine experimentation. Such equivalent items are intended to be encompassed in the scope of the claims herein.

Claims (1)

  1.   A method of modulating an immune response, comprising contacting an immune cell with an agent that modulates a signal through PD-1 and thereby modulating the immune response.
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